The results of molecular characterization of Hemophilia A in 50 patients from Southeast Bulgaria are presented. Southern blot analysis for the detection of inversions in intron 22, and polymerase chain reaction (PCR) followed by single strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE) for screening of the coding sequences of the Factor VIII (FVIII) gene were used. A molecular defect was found in 35 (70%), the most frequent being an inversion in intron 22, found in 19 (38%) patients; an intron 1 inversion was not detected. In one severely affected patient, an Alu insert was found, which disrupted exon 14 at codon 1224. Nucleotide substitutions were found in 15 (30%) patients, the most frequent being an Arg531→Cys missense mutation in exon 11. The same nonsense mutation (codon –5, CGA>TGA) was found in two patients with a severe phenotype. Seven missense mutations (Asn90→Thr, Arg 372→His, Glu456→Val, Tyr473→His, Arg1689→ Cys, Arg2159→Cys and Arg2163→His) were detected in isolated families. Two of these (Asn90→Thr and Glu456→ Val) are being reported for the first time.Pediatric Clinic, Medical Faculty, Stara Zagora, Bulgaria Key words: Hemophilia A; Factor VIII (FVIII) gene; Molecular characterization; Inversions; Point mutations