Besides having a role in signal transduction, heterotrimeric G proteins may be involved in membrane trafficking events. In chromaffin cells, Go is associated with secretory organelles and its activation by mastoparan inhibits the ATP-dependent priming of exocytosis. The effectors by which Go controls exocytosis are currently unknown. The subplasmalemmal actin network is one candidate, since it modulates secretion by controlling the movement of secretory granules to the plasma membrane. In streptolysin-O-permeabilized chromaffin cells, activation of exocytosis produces disassembly of cortical actin filaments. Mastoparan blocks the calcium-evoked disruption of cortical actin, and this effect is specifically inhibited by antibodies against Galphao and by a synthetic peptide corresponding to the COOH-terminal domain of Galphao. Disruption of actin filaments with cytochalasin E and Clostridium perfringens iota toxin partially reverses the mastoparan-induced inhibition of secretion. Furthermore, the effects of mastoparan on cortical actin and exocytosis are greatly reduced in cells treated with Clostridium botulinum C3 exoenzyme, which specifically inactivates the small G protein Rho. We propose that the control exerted by the granule-associated Go on exocytosis may be related to effects on the cortical actin network through a sequence of events which eventually involves the participation of Rho.