The function of rat, a ras-related GTP-binding protein, was investigated in fibroblasts by microinjection. In confluent serum-starved Swiss 3T3 cells, racl rapidly stimulated actin filament accumulation at the plasma membrane, forming membrane ruffles. Several growth factors and activated H-ras also induced membrane ruffling, and this response was prevented by a dominant inhibitory mutant rat protein, N17racl. This suggests that endogenous rat proteins are required for growth factor-induced membrane ruffling. In addition to membrane ruffling, a later response to both racl microinjection and some growth factors was the formation of actin stress fibers, a process requiring endogenous rho proteins. Using N17racl we have shown that these growth factors act through rat to stimulate this rho-dependent response. We propose that rat and rho are essential components of signal transduction pathways linking growth factors to the organization of polymerized actin.