Polyclonal rabbit anti-factor IX antisera were fractionated to establish solid-phase immunoassays recognizing calcium-dependent and non-calcium-dependent epitopes. The assays were greater than 99.9% specific for factor IX and sensitive to 0.05 U/dl plasma or 2 ng/ml purified factor IX. For the calcium-dependent fraction, an absolute requirement of divalent metal ions was found, and Sr(II), Mn(II), and Mg(II) could substitute for Ca(II). On immunoblots of reduced, electrophoresed factor IXa, the 125I-calcium-dependent antibody fraction bound to the amino-terminal light chain. Plasma sampled from 13 patients receiving warfarin and one with cephalosporin-related vitamin K deficiency had a mean level of calcium-dependent factor IX antigen of 22 U/dl, comparable to the 24 jU/dl average of factor IX procoagulant activity; these two results were highly correlated. Antigen levels determined by either the polyclonal or a monoclonal, non-calcium-dependent anti-factor IX assays ranged from 1.7-fold to 6.0-fold greater than the corresponding levels of factor IX procoagulant activity or calcium-dependent antigen level for each subject's plasma. The difference reflects inactive, circulating factor IX. In contrast, factor IX antigen levels determined by an assay using a monoclonal, calcium-dependent anti-factor IX were from one half to one thirteenth as much as those measured by the polyclonal, calcium-dependent immunoassay. The disparity between results of calcium-dependent assays suggests that some Gla residues near the amino terminus of factor IX are relatively less important for normal procoagulant function of factor IX than others, are more sensitive to the effects of vitamin K antagonism or deficiency, and are important for the epitope recognized by this particular calcium-dependent, monoclonal antibody.