Both amidated gastrin (Gamide) and glycine-extended gastrin (Ggly) stimulate gastrointestinal cell proliferation and migration. Binding of Gamide to the cholecystokinin (CCK)-2 receptor activates small GTP binding proteins of the Rho family (Rho, Rac and Cdc42), and dominant negative mutants of Rho or Cdc42 block Gamide-stimulated cell proliferation and survival. In comparison little is known about the Ggly signalling transduction pathway leading to cell proliferation and migration. The present study has examined the roles of the small G proteins Rho, Rac and Cdc42 in Ggly-induced proliferation and migration of the mouse gastric epithelial cell line IMGE-5. Ggly stimulated the activation of Rho, and its downstream effector protein, ROCK. The activation of Rho and ROCK mediated Ggly-induced cell proliferation and migration as inhibition of Rho by C3, or ROCK by Y-27632, completely blocked these effects of Ggly. Ggly also stimulated tyrosine phosphorylation of focal adhesion kinase (FAK), and stimulation was reversed by addition of C3 and Y-27632. In contrast to the effects of Rho and ROCK, inhibition of the Rac or Cdc42 pathways by expression of dominant negative mutants of Rac or Cdc42 did not affect Ggly-induced cell proliferation and migration. These results demonstrate that Ggly stimulates IMGE-5 cell proliferation and migration through a Rho/ROCK-dependent pathway but not via Rac- or Cdc42-dependent pathways.