Vitronectin is a 75 kilodalton (kDa) cell-adhesive glycoprotein found in animal blood and connective tissue, also termed serum spreading factor, S-protein, and epibolin. It promotes attachment and spreading of animal cells on tissue culture dishes, and it also binds to collagen. We established four mouse hybridoma lines producing monoclonal antibodies (M1, M2, M4 and M5) to human vitronectin. By immunoblotting, both epitopes recognized by M4 and M5 were suggested to exist in the amino terminal 5 kDa portion of vitronectin, and both Ml and M2 bound to the adjacent 35 kDa portion. Cell spreading on vitronectin-coated dishes was inhibited by M4 = M5 > M1, but not by M2. Collagen binding to vitronectinwas inhibited by M2 >M4 =M5, but not by Ml. These results indicate that the collagen-binding site is located near the cell-binding site in the amino terminalhalf of vitronectin. Independent inhibition of vitronectin binding to the cell and to collagen by these monoclonal antibodies will provide a potential tool to dissect the structure and function of vitronectin.