Point mutations in the factor VIII gene are responsible for the majority of cases of hemophilia A, and only a small fraction of these mutations can be recognized by restriction endonuclease analysis. We have now used polymerase chain reaction and denaturing gradient gel electrophoresis to characterize single nucleotide substitutions in the factor VIII gene. Five regions of the gene were studied: exon 8, the 3' end of exon 14, exon 17, exon 18, and exon 24. A GC clamp was attached to the 5' PCR primer to allow detection of the majority of single base changes in DNA fragments ranging from 249 to 356 bp. Ten of eleven known point mutations were definitively separated. Fifty-two patients with unknown mutations were then studied by these methods, and the disease-producing mutation was found in three. First, we identified a new missense mutation in exon 14 which is the likely cause of hemophilia A in one patient (tyrosine changed to cysteine at amino acid residue 1709). Second, we found a new missense mutation in exon 18 in one patient (asparagine to aspartic acid at amino acid residue 1922). Third, a previously described mutation in exon 24 was detected (arginine changed to glutamine at amino acid residue 2209). In addition, a new polymorphic nucleotide substitution was found in intron 7. Moreover, these mutations can be detected when the GC-clamped PCR products from all five regions are run in the same denaturing gel. Our results indicate that denaturing gradient gel electrophoresis can be successfully applied to the analysis of point mutations in large genes whose transcripts are not readily available.