We have previously demonstrated regulatory roles for Rho subfamily of G-proteins in glucose- and calcium-induced insulin secretion. Herein, we examined regulation by these proteins of insulin secretion from betaTC3 cells elicited by mitochondrial fuels, such as the succinic acid methyl ester (SAME). Preincubation of these cells with Clostridium difficile toxin-B (200 ng/mL), which monoglucosylates and inactivates Cdc42 and Rac1, markedly decreased (> 70%) SAME-induced insulin secretion. Furthermore, exposure of betaTC3 cells to GGTI-2147 (20 microM), a selective inhibitor of the requisite prenylation of Rac1 and Cdc42, significantly reduced (> 80%) SAME-induced insulin release, suggesting that post-translational prenylation of these proteins is necessary for SAME-induced insulin release. Western blot analysis indicated localization of Cdc42, Rac1, and Ras in the beta cell mitochondrial fraction. Confocal microscopy revealed a modest, but inconsistent, increase in the association of either Rac1 or Cdc42 with Mitotracker, a mitochondrial marker, following exposure to SAME. These data suggest that activation of preexisting intramitochondrial Rac1 and Cdc42 may be sufficient to regulate SAME-induced insulin secretion. Together, our findings support a role for G-proteins in insulin secretion at a step dependent on mitochondrial metabolism. They also identify mevalonate-derived, isoprenoid modified Rho G-proteins as specific signaling molecules in recently proposed succinate mechanism of insulin release.