A sensitive solid phase enzyme immunoassay (EIA) was developed for the measurement of factor IX antigen (IX:AG), using rabbit antihuman factor IX antiserum and β-D-galactosidase, which enabled us to detect IX:AG as low as 10-4 U/ml. 37 patients with severe hemophilia B have been investigated by EIA, inhibitor neutralization assay and bovine brain prothrombin time. They could be divided into four genetic variants. 25% had normal levels of IX:AG but decreased levels of factor IX clotting activity. On crossed immunoelectrophoresis of the hemophilia B+ and hemophilia BM, we could not find abnormalities in electrophoretic mobilities compared to normal subjects in the presence of 1 mM Ca++ lactate.