Patients. Fifty unrelated patients with Hemophilia A, and members of their families, were studied. Severe and moderate phenotypes were observed in 40 and 10 of the patients, respectively. The clinical severity was determined according to standard criteria, based on the remaining FVIII activity [7]. Blood samples were obtained after informed consent. DNA was isolated from peripheral white blood cells using the classical phenol/chloroform extraction and the ethanol precipitation methods used in our laboratory [8]. Southern Blotting. The intron 22 inversion of the FVIII gene was analyzed by Southern blotting using the BclI restriction enzyme and a 0.9 kb EcoRI/ SstI fragment of plasmid p482.6 (ATCC), containing a part of the intron 22 homologous sequence. The probe was non radioactively labeled using the Fluorescein Gene Images Labeling and Detection System (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The same Southern blotting method was used for the determination of gross gene deletions or insertions in the FVIII gene. Genomic DNA was digested with the TaqI restriction enzyme and hybridized with probes A and B representing cDNA of the FVIII gene from exons 1-14 and 14-26, respectively [9]. Polymerase Chain Reaction (PCR), Single Strand Conformation Polymorphism (SSCP) and Denaturing Gradient Gel Electrophoresis (DGGE). Patients in whom an inversion in intron 22 was not found to be a cause of Hemophilia A, were further tested for the presence of single nucleotide changes or small deletions/ insertions in the coding regions of the FVIII gene. The intron 1 inversion was detected using two sets of PC reactions according to Kemball-Cook et al. [5]. All exons,except exon 14, were separately amplified from genomic DNA by PCR using oligonucleotide primers and cycling conditions as described by David et al. [10] for SSCP or by Diamond et al. [11] for DGGE. The SSCP and DGGE tests were performed on the DeCode System (Bio-Rad Laboratories, Hercules, CA, USA). DNA Sequencing. To identify the nucleotide substitutions responsible for altered electrophoretic mobility detected by SSCP or DGGE analyses, each of the PCR fragments was sequenced, using the Big- Dye Terminator v1.1 kit (PE Applied BioSystems, Foster City, CA, USA) on an ABI PRISM™ 310 Genetic Analyzer (PE Applied Bio Systems). All sequence changes were confirmed in both strands, and analyzed by alignment with normal sequences in the database.