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A panel of human tumor cell lines was screened for selective expression of laminin ␣5 chain, a newly identified laminin subunit comprising laminin-10 (␣5␤1␥1) and -11 (␣5␤2␥1). The lung adenocarcinoma cell line A549 was found to express the ␣5 chain at relatively high levels but no detectable amounts of other ␣ chains. The laminin variants containing ␣5 chain were purified from the conditioned medium of A549 cells by immunoaffinity chromatography using the anti-laminin monoclonal antibody 4C7 which was shown recently to recognize the laminin ␣5 chain (Tiger, C.-F., Champliaud, M.-F., Pedrosa-Domellof, F., Thornell, L.-E., Ekblom, P., and Gullberg, D. (1997) J. Biol. Chem. 272, 28590 -28595). The purified laminin variants consisted of three chains with molecular masses of 350, 220, and 210 kDa. The 350-kDa chain was specifically recognized by another anti-␣5 chain monoclonal antibody capable of recognizing denatured ␣5 chain on immunoblots, whereas the 210-kDa chain was recognized by an anti-␥1 chain antibody. The purified ␣5 chain-containing laminin variants (hereafter referred to as laminin-10/11) were highly active in mediating adhesion of A549 cells to the substratum with potency as high as that of laminin-5 and significantly higher than those of laminin-1, laminin-2/4, or fibronectin. Adhesion to substrata coated with laminin-10/11 was specifically inhibited by anti-integrin antibodies directed against the integrin ␣3 or ␤1 subunit but not by those against ␣2 or ␣6 subunit, indicating that laminin-10/11 is specifically recognized by integrin ␣3␤1. Given the wide distribution of laminin-10/11 in the basement membrane of various tissue types and dominant expression of integrin ␣3␤1 in most epithelial cells, specific interaction of laminin-10/11 with integrin ␣3␤1 may play an important role in in vivo regulation of proliferation and differentiation of epithelial cells through the basement membrane.