A gross deletion in the factor VIII (FVIII) mRNA was determined by reverse transcriptase polymerase chain reaction (RT-PCR) for a patient with moderately severe hemophilia A. Sequencing of the RT-PCR product depicted a 177-bp deletion ranging from nucleotide (nt) 6724 to nt 6900 of FVIII cDNA, exactly corresponding to the whole exon 25. Further study of the genomic DNA revealed the presence of a single base pair substitution (G >A) at position -1 of intron 24. The absolute consensus AG doublet of the intron 24 splicing acceptor changed to AA. In the novel splice site mutation, exon 24 was erroneously spliced to exon 26, skipping exon 25. The FVIII antigen level was normal despite the markedly reduced functional activity. Since exon 25 corresponds to part of the C2 domain, we speculate that for this patient the aberrant C2 domain markedly reduces binding affinity of FVIII protein to the phospholipid membrane, thus severely impairing the protein function.