Two men with factor IX (FIX)antigen-positive (CRM+) hemophilia B were selected for study because of their abnormal expression of an immunologically defined epitope previously localized to the EGF-like domains of the molecule. Exons IV and V (coding for the first and second EGF-like domains) of FIX were amplified 10(7) times from the patients' genomic DNA by using polymerase chain reaction (PCR) technology and sequenced. Both patients had identical mutations which resulted in the highly conserved Gly 60 residue being changed to Ser. PCR-amplified exon IV from six normal males had the previously defined canonic sequence. The correlation between the mutation and defective epitope expression in the two patients suggests that a change in the tertiary structure of the EGF-like domain is likely to cause the mild hemophilia B.