The precise quantitation of activated factors in human factor IX concentrates has been accomplished with the use of recently developed, specific assays for factors IXa, Xa, and thrombin. The assay for factor IXa, which measures the initial rate of 3H-factor-X activation, was shown to be specific for factor IXa in the concentrates. Activated factor IX concentrates contained 1.0–2.3 microgram/ml of factor IXa; whereas the assays of unactivated concentrates were negative (less than 0.2 microgram/ml). The assays of factor Xa and thrombin, which measure the initial rate of p-nitroaniline release from S-2222 and S-2238, respectively, showed similar small amounts of factor Xa (4–34 ng/ml) and thrombin (12–76 ng/ml) in the activated and unactivated concentrates. The nonactivated partial thromboplastin time of the concentrates correlated significantly with the factor IXa content, but not with factor Xa or thrombin. Antithrombin III antigen in 3 of 4 concentrates was several-fold higher than antithrombin III activity, suggesting the presence of antithrombin III complexed with activated factors. These results support the hypothesis that the degree of activation of factor IX concentrates is related primarily to the concentration of factor IXa, which may be responsible for the thrombogenicity of these concentrates in some clinical settings.