Monoclonal antibodies were prepared against a cell attachment-promoting protein, serum spreading factor, which had been partially purified from human serum by chromatography on glass bead columns. The antibodies selected were those that reacted with polypeptides that had cell attachment-promoting activity after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunochromatography of human plasma on columns containing the monoclonal antibodies followed by affinity chromatography on heparin-Sepharose yielded material that in sodium dodecyl sulfate/polyacrylamide gel electrophoretic analysis gave polypeptides of molecular mass 65 and 75 kilodaltons. Both polypeptides bound each of three monoclonal antibodies and had cell attachment-promoting activity after transfer to nitrocellulose filters. Immunofluorescent staining of tissues with the monoclonal antibodies revealed a fibrillar pattern that was mostly associated with loose connective tissue and overlapped with fibronectin fibrils. Fetal membrane tissue, which showed strong staining with the antibodies in immunofluorescence, also gave 65- and 75-kilodalton polypeptides with cell attachment-promoting activity after chromatography on columns containing the monoclonal antibodies. One source of the tissue protein may be fibroblastic cells, because cultured human fibroblasts also stained with the monoclonal antibodies. The staining was fibrillar and appeared to be associated with the cell surface extracellular matrix. We propose the name "vitronectin" for the various forms of this protein, on the basis of its binding to glass and its adhesive properties.