Rat myotubes cultured in fetal calf serum adhere to vitronectin-coated substrates through two distinct structures, focal contacts and clathrin-coated membrane domains. We studied the integrins in myotubes to learn how they associate with these two domains. Double label immunofluorescence studies with antibodies specific for clathrin, vinculin and several forms of integrin showed that focal contacts and clathrin-coated membrane domains contain both vitronectin receptors (VnR, containing beta-3 and beta-5integrins) and fibronectin receptors (FnR, containing beta1-integrin). VnR but not FnR associates tightly with the substrate in both domains, as the VnR alone remains attached to the coverslip when the lipid bilayer and other membrane proteins are removed by detergent. Ultrastructural studies confirmed the localization of the beta5 subunit of the VnR at both domains. We used intracellular injection and affinity chromatography to test the possibility that clathrin at coated membrane domains associates with the cytoplasmic sequence of the beta5 subunit of the VnR. Injection of a synthetic peptide containing the NPXY motif from the cytoplasmic domain of the human beta5 subunit, SRARYEMASNPLYRKPIST, depleted clathrin from coated membrane domains without affecting clathrin in perinuclear structures or vinculin at focal contacts. Injection of the homologous beta1 peptide, MNAKWDTGENPIYKSAVITT, also containing an NPXY motif, had no significant effect on any of these structures. Affinity matrices containing the beta5 but not the beta1 peptide selectively retained clathrin from myotube extract, and bound clathrin could be selectively eluted by soluble forms of the beta5 but not the beta1 peptide. Thus, a sequence including the NPXY motif in the integrin beta5 subunit is involved in the specific anchoring of the VnR, but not the FnR, to clathrin-coated membrane.