Cell migration on fibronectin (FN)-coated substrata was studied using 10 cell lines, of which only 2 showed clear enhancement and 1 showed marginal enhancement of cell migration. The migration of the other 7 cell lines was not affected on FN-coated substrata, although they all showed FN-dependent cell adhesion. The migration-enhancing activity of FN was found in the fragment including the cell-adhesion and Hep-2 domains, but not other domains (Hep-1/Fib-1, Gel, Fib-2). No difference in the migration-enhancing effect was seen among FNs from plasma, fibroblasts, or transformed cells. FN-dependent cell migration was inhibited by polyclonal antibodies directed to the C-terminal half region including the cell binding domain, but not by antibodies directed to five other domains. Since these results indicated that FN-mediated cell migration could be controlled by the cell-adhesion domain of FN and its receptor, studies were then focused on the effect of antibodies directed to receptors for FN and collagen, and on the effect of tetrapeptide sequences recognized by these receptors. It was found that (i) cell migration on FN-coated surfaces was specifically inhibited by anti-FN receptor antibody P1F8 but not by anticollagen receptor antibody P1H5; (ii) the migration was strongly inhibited by Arg-Gly-Asp-Ser but not by other oligopeptide sequences. However, the majority of those cell lines not susceptible to FN-dependent cell migration were characterized by having FN receptors and the ability to adhere on FN-coated matrix. Based on these findings, it was concluded that FN-dependent cell migration shares the same recognition mechanism as FN-dependent cell adhesion, but that the majority of cell lines not exhibiting FN-dependent migration still show FN-dependent cell adhesion and express the FN receptor (integrin); i.e., cell migration and adhesion involve the same receptor and the same FN loci, but migration is controlled by still-unidentified cellular factors which determine the susceptibility of the cell to the dynamic function of the FN receptor (integrin) unit.