The interaction of the heparin-dependent antibody with heparin and platelets has been studied using the sera and purified IgG of four patients with heparin-induced thrombocytopenia. Both normal platelets and Bernard-Soulier syndrome (BSS) platelets which lack glycoprotein (GP) Ib, GPV and GPIX, aggregated in response to patient serum or IgG, but only in the presence of heparin. A monoclonal antibody (Mab) against platelet Fc II receptor (IV. 3) strongly inhibited the heparin-dependent aggregation of both normal and BSS platelets induced by patient sera/IgG. Inhibition by the anti-GPIb Mab (AK2) was variable and occurred only with normal platelets. Anti-GPIX Mab (FMC 25) was not inhibitory with either normal or BSS platelets. Similar results were obtained using 14C-serotonin release instead of platelet aggregation as a measure of platelet activation. These findings suggest that (1) the reaction of the heparin-dependent antibody with platelets and heparin is mediated by a Fc-dependent mechanism, (2) GPIb, GPV and GPIX are not involved in this reaction, and (3) the inhibitory effect of anti-GPIb Mab on normal platelets is due to steric interference consistent with the platelet Fc receptor being in close proximity to GPIb.