Binding of Staphylococcus aureus to vascular endothelial cells may be an initiating event in the pathogenesis of endovascular infection, particularly infective endocarditis. In this study a competition assay between labeled and unlabeled bacteria was used to identify potential S. aureus-binding determinants on the surface of cultured porcine aortic valve endothelial cells. Concanavalin A inhibited 30% to 40% of the specific binding of S. aureus to membrane-associated components. Concanavalin A affinity chromatography of radiolabeled cell-surface proteins, followed by isolation of S. aureus-binding proteins, identified a 130,000-molecular-weight cell surface protein that may function as an endothelial cell S. aureus receptor. These data suggest that specific binding of S. aureus to cardiac valve endothelial cells is mediated in part by a 130 kd mannose-containing glycoprotein.