The GTP-binding proteins Rho, Rac and Cdc42 are known to regulate actin organization: Rho induces the assembly of contractile actin-based filaments such as stress fibres, Rac regulates the formation of lamellipodia and membrane ruffles, while Cdc42 is required for filopodium extension. All three proteins can also regulate the assembly of integrin-containing focal adhesion complexes. Cell migration involves co-ordinated and dynamic changes in the actin cytoskeleton and cell adhesion, and we have therefore investigated the roles of Rho family proteins in migration, using two model cell systems. First, in the macrophage cell line Bac1, Rho and Rac were found to be required for colony-stimulating factor-1 (CSF-1)-induced cell migration. In contrast, inhibition of Cdc42 does not prevent macrophages migrating in response to CSF-1, but does prevent recognition of a CSF-1 concentration gradient, so that cells now migrate randomly rather than up the gradient. This implies that Cdc42, and probably filopodia, are required for gradient sensing and cell polarization. Secondly, in the Madin-Darby canine kidney (MDCK) epithelial cell line, Rho and Rac are also essential for migration induced by hepatocyte growth factor/scatter factor. Rac is required for lamellipodium formation and is apparently activated via Ras. Interestingly, however, Rac does not induce lamellipodium formation in unstimulated MDCK cells, indicating that Rac signals differently in epithelial cells compared with fibroblasts or macrophages. Our results point to central roles for Rho, Rac and Cdc42 in co-ordinating cell migration.