A radioimmunoassay for alloantibodies (inhibitors) and heteroantibodies to human factor IX has been developed using radioiodinated human factor IX and formalin-fixed, heat-killed Staphylococcus aureus cells (Staph A). Staph A was used as a solid-phase adsorbent for immune complexes. The assay is specific, shows excellent correlation with factor IX coagulant neutralization assays in detecting alloantibodies (r = 0.98), and is 60 times more sensitive. The Staph A method allows rapid separation of immune complexes (within 10 min of addition) and binds immunoglobulin with equivalent efficiency in the presence and absence of calcium. These characteristics have made the Staph A binding method useful for observing the Ca++ effect on the antigenicity of factor IX as detected by hetero- and alloantibodies. All antisera investigated showed some increase in antibody titer when measured in the presence of Ca++. One particular alloantibody showed the greatest increase in titer (> twofold). The reversibility of the calcium effect by excess EDTA indicates that it was not caused by Ca++-dependent proteolysis of the factor IX molecule. The Staph A procedure can also be used as a sensitive competitive radioimmunoassay for human factor IX using alloantibody. The assay could detect factor IX antigen to a dilution of 1:1280 of normal plasma and correctly classified hemophilia B plasmas as cross-reacting material positive or negative.