In isolated rat pancreatic acini, Src, RhoA, PI3-K, Vav-2, G α12 , and G α13 were detected by immunoblotting. CCK enhanced the levels of these proteins, and the levels of Src and RhoA were reduced by the Src inhibitor herbimycin A and the Rho inhibitor pravastatin. The PI3-K inhibitor wortmannin reduced the level of PI3-K. These inhibitors also decreased amylase secretion in CCK-treated pancreatic acini without altering basal secretion. Immunoprecipitation studies indicated that CCK caused Src to associate with Vav-2, RhoA, and PI3-K and RhoA and Src to associate with Vav-2. Ras, RasGAP, and SOS did not coimmunoprecipitate with Vav-2, and RasGAP and SOS did not coimmunoprecipitate with RhoA. CCK also enhanced Vav-2 and RhoA to coimmunoprecipitate with G α13 . We conclude that CCK stimulates the recruitment of the Src–RhoA–PI3-K signaling pathway by Vav-2 downstream of G α13 in pancreatic acini.