Tumor cell adhesion to the triple-helical domain of basement membrane (type IV) collagen occurs at several different regions. Cellular recognition of the sequence spanning α1(IV)531-543 has been proposed to be independent of triple-helical conformation (Miles, A. J., Skubitz, A. P. N., Furcht, L. T., and Fields, G. B. (1994) J. Biol. Chem. 269, 30939-30945). In the present study, integrin interactions with a peptide analog of the α1(IV)-531-543 sequence have been analyzed. Tumor cell adhesion (melanoma, ovarian carcinoma) to the α1(IV)531543 chemically synthesized peptide was inhibited by a monoclonal antibody against the α3 integrin subunit, and to a lesser extent by monoclonal antibodies against the β1 and α2 integrin subunits. An anti-α5 monoclonal antibody and normal mouse IgG were ineffective as inhibitors of tumor cell adhesion to the peptide. Two cell surface proteins of 120 and 150 kDa bound to an α1(IV)531-543 peptide affinity column and were eluted with 20 mM EDTA. When the eluted proteins were incubated with monoclonal antibodies against either the α3 or β1 integrin subunit, proteins corresponding in molecular weight to α3 and β1 integrin subunits were precipitated. No proteins were immunoprecipated with monoclonal antibodies against the α2 or α5 integrin subunits. Thus, the α3β1 integrin from two tumor cell types has been shown to bind directly to the α1(IV)531-543 peptide. The α1(IV)531-543 peptide is the first collagen- like sequence that has been shown to bind the α3β1 integrin.