The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because (1) it immunoprecipitated drug-dependently the former but not the latter glycoprotein complex and (2) the MAIPA assay showed strong rifampicin-dependent antibody binding when anti-GPIb-IX monoclonal antibodies (mAbs) (AK2 and FMC25) but not anti-GPIIb-IIIa mAbs (AP2, SZ21, and SZ22) were used to capture the antigen. The antibody binding site was further localized to the GPIX subunit of the GPIb-IX complex because flow cytometric analysis revealed drug-dependent antibody binding to L cells transfected with human GPIbbeta and GPIX complementary DNA (L betaIX cells) but not with human GPIbalpha and GPIbbeta complementary DNA (L alphabeta cells). Finally, in the MAIPA assay, the rifampicin-dependent antibody almost completely cross-blocked the binding of the anti-GPIX mAb (SZ1) to platelets. Similar cross-blocking of SZ1binding to platelets by the quinine-dependent antibodies was also observed. This finding not only confirms that the epitope of the rifampicin-dependent antibody is on GPIX but it is also identical to or located in close proximity to that of the quinine-dependent antibody and SZ1. Further characterization of the epitopes of these antibodies may have important implications for a general understanding of the mechanism of drug-induced thrombocytopenia. (Blood. 2000;95:1988-1992)