Staphylococcus saprophyticus, an important cause of urinary tract infections, produces two major surface proteins, the S. saprophyticus surface-associated protein (Ssp) and the hemagglutinin, which mediates fibronectin binding and also functions as the major adhesion of the organism. The hemagglutinating and fibronectin binding functions probably reside on different parts of the molecule. To identify a receptor on eukaryotic cells, binding and inhibition studies with acidic and neutral glycosphingolipids, carbohydrates, and proteins of sheep erythrocyte membranes were conducted. S. saprophyticus did not bind to any glycosphingolipid and no inhibition was observed when hemagglutination assays were done in the presence of carbohydrates or fibronectin. Neither treatment of erythrocytes with galactose oxidase or neuraminidase and galactose oxidase nor mild periodate oxidation of erythrocytes reduced hemagglutination. However, proteinase-treated erythrocytes were no longer agglutinated. Similarly, untreated erythrocyte membranes inhibited hemagglutination, whereas proteinase-treated membranes did not. In addition, only hemagglutinating strains bound to 60- and 21-kDa sheep erythrocyte membrane proteins on ligand blots, and these proteins inhibited hemagglutination. Our data indicate that, in contrast to many other hemagglutinins, the receptor on sheep erythrocytes for S. saprophyticus is a protein.