A new murine monoclonal antibody, MDP-1, specific for human platelet glycoprotein IIIa has been produced and characterized. Following SDS-polyacrylamide gel electrophoresis, MDP-1 reacted with a 94kDa protein immobilized on a nitrocellulose membrane. Upon reduction, MDP-1 no longer bound to the 94kDa protein indicating an epitope requiring at least one disulfide bond. On crossed immunoelectrophoresis MDP-1 reacted to the same peak as the GP IIb-IIIa complex-specific antibody AP-2. After dissociation of the GP IIb-IIIa complex with EDTA, AP-2 showed no reactivity while MDP-1 bound to a new peak that was broader and anodal to the original GP IIb-IIIa peak, consistent with GP IIIa. MDP-1 inhibited ADP and thrombin induced aggregation. In addition, MDP-1 inhibited ADP induced release of ATP, but did not inhibit thrombin stimulated ATP release. Following chymotrypsin digestion, MDP-1 bound to a cleaved GP IIIa protein (nonreduced M, = 122 kDa) consistent with opening of the major disulfide loop. A second cleavage resulted in a 63 kDa species that reacted with MDP-1. Scatchard analysis revealed 22 000 molecules of MDP-1 bound per platelet, and indicated a type of binding consistent with positive cooperativity. The antibody bound equally well to stimulated and unstimulated platelets. MDP-1 binding was inhibited by a polyclonal anti-PIA1antibody, but bound to platelets from a PIA1negative individual indicating a binding site close to but not identical to the PIA1epitope. In addition, MDP-1 binding was not inhibited by Arg-Gly-Asp-Ser (RGDS) suggesting that it is not directed to the RGD binding site on GP IIIa.