Clinically used GPIIb/IIIa blockers are ligand mimetics, and thereby their binding can induce conformational changes of the platelet integrin GPIIb/IIIa. Since the reversibility of these conformational changes may be an important determinant of potential adverse effects of GPIIb/IIIa blockers, we produced a new monoclonal antibody (anti-LIBS-mAb), and by using its binding properties, we investigated the conformational changes of GPIIb/IIIa during the binding and especially the dissociation of GPIIb/IIIa blockers. Production of monoclonal antibody (mAb) clones was performed using purified GPIIb/IIIa in a high affinity conformation and using activated platelets. Clone anti-LIBS-145-mAb was chosen, since it allowed the sensitive probing of eptifibatide-induced conformational changes of GPIIb/IIIa. On resting and activated platelets and on GPIIb/IIIa-expressing Chinese hamster ovary cells, anti-LIBS-145-mAb binding returned to background binding after dissociation of eptifibatide, indicating a complete reversibility of the eptifibatide-induced conformational change. Furthermore, with the mixing of eptifibatide-preincubated and nonincubated cells, a fast reversibility could be demonstrated. However, when fibrinogen was present in a physiological concentration, the GPIIb/IIIa blocker-induced conformation was partially retained after the dissociation of eptifibatide and to the same extent binding of fibrinogen and the activation-specific mAb Pac-1 was induced. In conclusion, a fast reversibility of the conformational change of GPIIb/IIIa after dissociation of GPIIb/IIIa blockers could be demonstrated as an intrinsic property of the GPIIb/IIIa receptor. This mechanism prevents general platelet aggregation after dissociation of ligand mimetic GPIIb/IIIa blockers. Nevertheless, in the presence of fibrinogen this reversibility is not complete, which may explain some of the side effects of GPIIb/IIIa blockers, especially those of the oral GPIIb/IIIa blockers.