The polymerase chain reaction procedure (PCR) was used to detect a polymorphic Hha I site adjacent to the factor IX locus in a panel of 33 phenotypically normal caucasian individuals. This technique was also applied to a haemophilia B family pedigree. The Hha I polymorphic site was located 8 kb 3' to the factor IX gene, and the proportion of female subjects expected to be heterozygous at this site was 0.48. The Hha I locus was in linkage equilibrium with the other polymorphic loci on the factor IX gene. These findings, besides increasing the proportion of caucasian individuals whose haemophilia B carrier state can be diagnosed from 79% to 89%, demonstrate this widely applicable use of PCR for the detection of DNA polymorphism at cytosine phosphoguanadine dinucleotides irrespective of the methylation status.