Guanosine 5′- O-(3-thiotriphosphate) (GTPγS) activated the I Cl,swell anion channel in N1E115 neuroblastoma cells in a swelling-independent manner. GTPγS-induced current was unaffected by ATP removal and broadly selective tyrosine kinase inhibitors, demonstrating that phosphorylation events do not regulate G protein-dependent channel activation. Pertussis toxin had no effect on GTPγS-induced current. However, cholera toxin inhibited the current ∼70%. Exposure of cells to 8-bromoadenosine 3′,5′-cyclic monophosphate did not mimic the effect of cholera toxin, and its inhibitory action was not prevented by treatment of cells with an inhibitor of adenylyl cyclase. These results demonstrate that GTPγS does not act through Gαi/o GTPases and that Gαs/Gβγ G proteins inhibit the channel and/or channel regulatory mechanisms through cAMP-independent mechanisms. Swelling-induced activation of I Cl,swell was stimulated two- to threefold by GTPγS and inhibited by 10 mM guanosine 5′- O-(2-thiodiphosphate). The Rho GTPase inhibitor Clostridium difficile toxin B inhibited both GTPγS- and swelling-induced activation of I Cl,swell. Taken together, these findings indicate that Rho GTPase signaling pathways regulate the I Cl,swell channel via phosphorylation-independent mechanisms.