Although heparin induces immune-mediated thrombocytopenia, it has been difficult to demonstrate heparin specificity of the putative immunoglobin. Recently, however, a body of data has indicated that platelet factor 4 (PF4) is required for heparin-induced thrombocytopenia (HIT) antibody to bind to heparin. Using viable platelets in a physiologic buffer, we have now documented specific and reversible platelet binding of iodine 125-labeled IgG from 5 patients with HIT and binding of 125I-labeled F(ab')2 from 2 of them. The binding requires the presence of both heparin and PF4 in a molar ratio of approximately 1:1. We have also shown that platelet activation increases HIT antibody binding. Our data suggest that the F(ab')2 domain of HIT antibody binds to heparin-PF4 complexes that accumulate on the platelet surface when equimolar concentrations of heparin and PF4 are present.