We report here on gene transfer studies designed to investigate the function of the alpha 5 beta 1 integrin and its role in transformation. Transfection of the human alpha 5 and beta 1 cDNAs into transformed Chinese hamster ovary (CHO) cells followed by methotrexate-induced amplification yielded clonal cell lines overexpression this fibronectin receptor. The overexpressors deposited more fibronectin in their extracellular matrix and migrated less than control cells. In addition, they showed reduced saturation density and reduced ability to grow in soft agar. The overexpressor cells, unlike the control CHO cells, were nontumorigenic when injected subcutaneously into nude mice. The results indicate that extracellular matrix recognition by the alpha 5 beta 1 integrin plays a role in the control of cell proliferation and suggest that a reduction of this fibronectin receptor may be responsible for the acquisition of anchorage independence by transformed cells.