TO identify platelet surface structures involved in adhesion to collagen, the effect of 16 murine antiplatelet membrane hybridoma antibodies were tested in a defined, in vitro assay. Four of these antibodies inhibited platelet-collagen adhesion and reacted with a polypeptide with Mr ~ 125,000, as determined by immunoblots after gel electrophoresis under reducing conditions. Through detailed studies with one of these antibodies, the monoclonal antibody PMI-1, the relevant antigen was identified as platelet glycoprotein Ilba, based upon (a) co-migration with this glycoprotein in two-dimensional gel electrophoresis and (b) co-purification by immunoaffinity chromatography with a protein with apparent Mr identical to that of glycoprotein III, under conditions in which glycoproteins lib and III form a complex.Univalent antibody fragments prepared from monoclonal antibody PMI-1 inhibited >80% of platelet-collagen adhesion, and inhibition was completely blocked by the immunopurified antigen. These results indicate that glycoprotein lib participates in some aspect of plateletcollagen adhesion. In contrast, the purified antigen only partially neutralized a polyclonal antiserum that blocked platelet-collagen adhesion, to a maximum of ~25%, at saturating antigen concentrations. Thus, by these immunological criteria, glycoprotein lib is not the only molecule involved in this process.Antibodies are a major tool for identification of molecules that participate in cell adhesion. Iterative absorption of polyclonal antibodies has permitted identification of several cell adhesion molecules (1). An additional approach has employed monoclonal antibodies, which offer the advantage of exquisite immunologic specificity. These have been used to identify molecules involved in the adhesion of myoblasts (2) and melanoma cells (3) to surfaces in tissue culture, and of Dictyostelium discoideum cells to each other (4).In the preceding article, we described a polyclonal antiserum raised against whole human blood platelets that was employed to identify and partially purify neutralizing antigens involved in platelet-collagen adhesion (5). This work was, however, greatly complicated by the finding that the adhesion that was measured in this assay apparently involved several immunologically distinct molecular entities, each of which only partially neutralized the polyclonal antiserum. Because of the complexities of the system, we turned to monoclonal antibodies to develop monospecific reagents that might react with the relevant entities individually, permitting their clear identification and independent characterization. We describe here a monoclonal antibody that reacts specifically with platelet membrane glycoprotein IIb and blocks platelet-collagen adhesion. The platelet antigen purified by immunoaffinity chromatography, using immobilized monoclonal antibody, only partly neutralizes the polyclonal antiserum described in the preceding article (5), supporting the conclusion that multiple entities are involved in platelet-collagen adhesio...