We investigated the involvement of Rho GTPases in the secretory process of PC12 cells. Overexpression of wild-type RhoA, Rac1, or Cdc42 did affect exocytosis. In contrast, secretion elicited by depolarizing K(+) concentrations was enhanced by the dominant negative mutants RhoA(N19), Rac1(N17), and Cdc42(N17) and was diminished by the constitutively active mutants RhoA(V14), Rac1(V12), and Cdc42(V12). The inhibition observed in the presence of RhoA(V14) was likely a result of the activation of ROK(alpha), since the catalytic domain of this kinase was able to mimic both the reorganization of the actin cytoskeleton and the decrease in exocytosis induced by the RhoA mutant. Part of the effect of Rac1(V12) may be due to POR1 activation. Thus, overexpression of full-length POR1 diminished K(+)-stimulated exocytosis, and a point mutation in the effector domain of Rac1(V12) that prevents the interaction with POR1 abolished the inhibitory effect of the GTPase. We also searched for the Cdc42(V12) target but overexpression of the Cdc42 effector WASP did not mimic the inhibition of exocytosis observed in cells transfected with the activated GTPase. Our findings indicate that different signaling cascades resulting in the activation of RhoA, Rac1, or Cdc42 can modulate the exocytotic process of neuroendocrine cells.