Hemophilia B is due to multiple molecular defects in the factor IX gene. Over 80% of mutations are single base substitutions. By amplification and direct sequencing, 51 single base substitutions were found in the transcribed sequence of the factor IX genes of patients from 50 distinct families with hemophilia B. These include 30 mutations in 29 families not previously reported by us; of these, 12 are novel, i.e., not previously published in other series. Of the 51 substitutions in our overall series 23 (45%) occurred as C-to-T or G-to-A transitions at 11 sites within CG dinucleotides. It is estimated that CG transitions occur from one to two orders of magnitude more frequently than mutations in nucleotides that are not within a CG pair. More than one family had identical defects for 6 of the CG mutations. At 4 of these sites, most patients had different haplotypes compatible with distinct mutations. Non-CG-type mutations occurred throughout the coding regions with only one mutation in more than one family. The latter included 7 families with a 397 Ile-to-Thr defect that all share a rare haplotype, suggesting a common ancestor.