In situ hybridization is a valuable molecular method that permits evaluation of the results in the context of preserved cellular morphology. It was introduced and standardized in our department almost two decades ago. The working protocol is simple to perform and results are avail able in less than 24 hours. In comparison to other molecular methods for detection of HPV DNA, ISH has many advantages. In contrast to polymerase chain reaction (PCR), expensive equipment is not needed, and the risk of contamination of samples is avoided. The major disadvantage of CISH is its limited sensitivity. To detect the presence of one or a few HPV DNA copies in invasive carcinoma cells, we chose the method of catalyzed signal amplification ISH as the most appropriate. Our study was performed on archival material (5-13 year-old formalin-fixed paraffin embedded tissue samples). Even though archival material could present problems with non specific staining, the age of the paraffin blocks did not significantly influence the results we obtained, as reported by others [25,30] before our study was made. In order to adapt the method of ISH TSA to the conditions in our department, some modifications in the working protocol were necessary. We achieved an optimal balance between intensity of signal and preservation of tissue morphology by prolonging the pretreatment time for 10 min. and increasing the temperature by 5°C, thus increasing the permeability of the cellular membrane with no effect on the tissue morphology. When using biotinylated probes, the non specific background staining, caused by the presence of endogenous streptavidin binding sites in the tissues, can be a serious problem [31]. In our study, the increased non specific background staining occurred only in small biopsies and mucin-producing neoplasms, but except in one case, did not significantly interfere with the specific staining. With CISH, we detected HPV DNA in 33.7% cases of early stage invasive cervical carcinoma. However, using ISH TSA, the sensitivity of CISH was increased by 24.7%, and even multiple HPV infections were detected. Several studies have compared hybridization techniques for detection of HPV DNA [22,32-34]. For example, Lizard et al. [32] evaluated the catalyzed signal amplification system for the ISH using biotinylated DNA probes on CaSki, SiHa, and HeLa cells and found the GenPoint (DAKO) method to be superior to immuno-enzymatic procedures, easily allowing detection of 1-2 copies of HPV DNA in the isolated cells. Wiedorn et al. [33] compared the sensitivity of the CISH, ISH TSA and in situ PCR (direct and indirect) methods on 100 cervical biopsies with precursor lesions and concluded that ISH TSA has sensitivity similar to that of the in situ PCR method. In a similar study [34] that compared the sensitivity of CISH, ISH TSA and in situ PCR, on HeLa, CaSki cells and 30 biopsies from precursor cervical lesions, the sensitivity of ISH was increased by 6.6-13.3% in lowgrade squamous intra epithelial lesions (LSIL) and high-grade squamous intra epithelial lesions (HSIL), when the ISH TSA step was added to the protocol. The authors of that study hypothesized that, when detecting HPV DNA on archival material, ISH and conventional PCR methods are compatible and are not mutually exclusive. Poddighe et al. [6] tested SiHa, HeLa, CaSki cells and 20 cervical biopsies containing normal epithelium, precursor lesions or invasive carcinomas for the presence of HPV DNA using the CISH, ISH with TSA and PCR. As in our study, these authors detected 20% improvement in sensitivity of the ISH with the TSA step. Sano et al. [22] tested 28 cases of cervical carcinomas and 53 CIN lesions for the presence of HPV type 16, using the ISH TSA method. Eighteen of 28 carcinomas (64%) contained HPV DNA type 16. However, in our study, the percentage of HPV type 16/18 positive carcinomas was lower (46.8%), probably due to the differences in the case selection. The presence of multiple types of HPV is not rare in cervical carcinomas. According to the literature, multiple HPV infections occur in 5-10% of invasive cervical carcinomas [35,36]. The most common types in multiple infections are HPV types 16/18 [35,36]. According to Bosch et al. [37], multiple HPV infection can be encountered in 20% of invasive cervical carcinomas, but only when PCR and ISH were used concomitantly. In our study, multiple HPV infections were detected in 8.9% of the cases. All the above mentioned studies suggest that ISH TSA is superior to CISH when it comes to detection of HPV in cervical lesions that contain only few copies of the viral DNA, such as invasive cervical carcinomas. As suggested by others [6,32-34], the added step of TSA should increase the sensitivity of conventional ISH by up to 25%. Although we used a highly sensitive ISH method for detection of HPV DNA, we found no HPV in 40.3% of our cases. This may be explained by infection with types of HPV for which probes were not included, by sampling error, by loss of the HPV during the tumor progression, or because the tumor was not HPV-associated. Sampling error could explain the occurrence of the one case in which presence of HPV types 16/18 was detected with CISH but could not be confirmed with ISH TSA. A major advantage of ISH is the possibility to estimate the physical state of HPV DNA. With ISH TSA, we detected 20 new HPV-positive cervical carcinomas that had dot hybridization signal (one or a few copies of possibly integrated HPV DNA). Similar results were reported in a comparison of the sensitivity of CISH and ISH TSA on SiHa, HeLa and CaSki cells, and 17 samples from genito-urinary tract, using GenPoint kit from DAKO [38]. Unger et al. [28 ] tested 47 cervical carcinomas for the presence of HPV DNA using CISH and PCR and found integrated HPV DNA in 77% of their cases. In situ hybridization with TSA was also superior to CISH in detection of a higher number of positive cells per sample. Accordingly, detection of more than five positive cells per tissue sample was increased by 9% when the TSA step was added. Our results compare with those reported by others [20,25]. In three of the cases in our study, the number of infected cells was higher when CISH was used. This conflicting result was probably caused by a sampling error, namely, in the preparation step, tissue sections were cut in different levels, which presumably contained cells with a different number of viral DNA copies. In conclusion, we found ISH TSA to be a fast and simple method for detection of HPV DNA in tissues containing only one or a few copies of HPV DNA, such as in early stage invasive cervical carcinoma. The addition of the TSA step increased significantly the sensitivity and the efficiency of the CISH in detection of the presence of HPV. The ef- ficiency of ISH TSA was also higher in detection of the HPV type, number of positive cells and assessment of the physical state of HPV DNA.