A circulating inhibitor specific for factor IX in a patient with hemophilia B was characterized with antisera to human immunoglobulins. Inhibitor-rich plasma was mixed with an excess of specific antiserum and then assayed for residual inhibitor activity. Inhibitor activity was completely removed by antisera specific for γG4 heavy chains and lambda light chains. Antisera specific for γA, γM, γD, γE, γG1, γG2, and γG3 heavy chains and kappa light chains had no effect on the inhibitor. On preparative zone electrophoresis, inhibitor activity was localized in a sharp band in the anodal portion of the γG peak, an electrophoretic distribution paralleling that of γG4. Precise localization of the inhibitor activity on calibrated gel filtration columns revealed a relatively narrow zone of fractions containing the inhibitor, wherein the proteins have an estimated molecular weight of 145,000. The relative homogeneity of the antibody may reflect specificity for a uniform, discrete portion of the human factor IX molecule.