Most heterotrimeric G protein alpha subunits are covalently modified by palmitate attached to one or more N-terminal cysteine residues. Although a wide variety of proteins undergo palmitoylation, the role of this fatty acid modification in G protein signaling is not well understood. Thus, we examined the role of palmitoylation of alpha(13), a G protein alpha subunit that regulates many pathways involved in cell growth. Both N-terminal cysteines at positions 14 and 18 were required for palmitoylation. Mutant alpha(13), in which both cysteines were changed to serines, failed to localize to plasma membranes in transfected cells and failed to activate Rho-dependent serum response factor-mediated transcription and actin stress fiber formation. However, nonpalmitoylated, cysteine to serine mutant alpha(13) retained the ability to co-immunoprecipitate with a direct effector, p115-RhoGEF. Finally, we report the novel observation that activated alpha(13) induces a redistribution of p115-RhoGEF from the cytoplasm to plasma membranes, but non-palmitoylated mutants of alpha(13) fail to cause p115-RhoGEF translocation. These findings identify palmitoylation of alpha(13) as critical for its proper membrane localization and signaling and provide insight into the mechanism of activation of Rho-dependent signaling pathways by alpha(13).