In chronic immune thrombocytopenic purpura (ITP), autoantibodies bind to platelet surface proteins, particularly alphaIIb, resulting in platelet destruction by the reticulo-endothelial system. In order to better localize the autoepitopes on alphaIIb, we studied the binding of antibodies to Chinese hamster ovary (CHO) cells expressing either alphaIIbbeta3 or alphaIIb-alphavbeta3 chimaeras in which a segment of alphaIIb (either amino acids L1-Q459, L1-F223 or F223-Q459) was substituted for that portion of alphav. We evaluated platelet-associated autoantibodies from 14 ITP patients with alphaIIb-dependent antibodies. Ten of 14 bound to alphaIIb (L1-Q459)-alphavbeta3, showing that autoepitopes were often localized to this region of alphaIIb. In addition, each of the autoantibodies binding to alphaIIb (L1-Q459)-alphavbeta3, also bound to CHO cells expressing either alphaIIb(L1-F223)-alphavbeta3 or alphaIIb(F223-Q459)-alphavbeta3). In two of the three eluates tested, > 95% of the autoantibody binding to alphaIIb could be adsorbed using CHO cells expressing any of the three chimaeras, showing that the epitope(s) have contact points on either side of amino acid F223; in the third eluate, only a portion ( approximately 40%) could be adsorbed by the chimaeric cell lines showing that, in this patient, an additional antibody was also present, directed to a site distal to amino acid Q459. The remaining four eluates bound to CHO cells expressing alphaIIbbeta3 but to none of the chimaeras, suggesting that these epitopes are also distal to amino acid Q459. We conclude that the binding of many anti-alphaIIbbeta3 autoantibodies is dependent on the presence of alphaIIb amino acids L1-Q459.