BACKGROUND: Hemophilia A is the most common X-linked bleeding disorder with an incidence of 1/5,000 males. Inversions within the factor VIII gene cause almost half of all cases of severe hemophilia A. However, DNA-based diagnosis has previously been carried out only by linkage analysis in Korean hemophilia A families. In this study, we aimed to establish direct inversion detection using a single-tube polymerase chain reaction (PCR) assay. METHODS: We have modified a single-tube PCR assay that combines overlapping PCR with long-distance PCR; performing PCR directly from genomic DNA with four primers P, Q, A, and B that differentiate the wild type, inversion, and the carrier detected the inversion. RESULTS: Segments PQ (12 kb) and AB (10 kb) were produced in hemizygous wild-type males. Males with hemophilia A due to the inversion showed segments PB+AQ (11 kb) along with the 10 kb segment from the nonrecombined extragenic homologue. In 20 (18.7%) patients, an inversion was found. The three segments were readily identifiable and all PCR amplifications achieved uniform reproducible results. CONCLUSIONS: The PCR was successful for the direct detection of factor VIII gene inversions. The method is simple, inexpensive, and more standardized; therefore, it may be the natural starting point for ascertaining mutations in families with severe hemophilia A.