Chronic immune thrombocytopenic purpura (ITP) is a syndrome of destructive thrombocytopenia due to autoantibodies against platelet-associated antigens. These antigens are most commonly located on the platelet glycoprotein (GP) IIb/IIIa complex. In the present studies, we show that many platelet-associated anti-GPIIb/IIIa autoantibodies from chronic ITP patients depend on conformationally intact GPIIb/IIIa for maximal binding. We studied anti-GPIIb/IIIa autoantibodies from 19 ITP patients (15 platelet-associated, 8 plasma) and alloantibodies from three patients with posttransfusion purpura (anti-PIA1). Antibodies were preincubated with purified intact GPIIb/IIIa, EDTA-dissociated GPIIb/IIIa, GPIIIa, or GPIIb for 2 hours and then residual antibody was measured in an antigen capture assay. The binding results were compared with those obtained using antibody preincubated in buffer. Of the 15 platelet-associated autoantibodies studied, the intact GPIIb/IIIa complex resulted in greater inhibition of antibody binding than the EDTA-dissociated complex, with a mean inhibition ratio (intact/dissociated) of 7.9 (range, 1.4 to 30.3). Little inhibition was noted using either GPIIb or GPIIIa. Conversely, plasma anti-PIA1 alloantibodies or plasma autoantibodies from ITP patients against the c-terminal region of GPIIIa were more efficiently inhibited by the dissociated complex or purified GPIIIa. We conclude that platelet-associated anti-GPIIb/IIIa autoantibodies in chronic ITP are frequently directed to cation-dependent conformational antigens.