Mitochondrial Ca²⁺ handling has been implicated in spontaneous rhythmic activity in smooth muscle and interstitial cells of Cajal. In this investigation we evaluated mitochondrial inhibitors on spontaneous action potentials (APs), Ca²⁺ flashes and Ca²⁺ waves in gallbladder smooth muscle (GBSM). Disruption of the mitochondrial membrane potential with CCCP, FCCP, rotenone, and antimycin A significantly reduced or eliminated APs, Ca²⁺ flashes and Ca²⁺ waves in GBSM. Blockade of ATP production with oligomycin did not alter APs or Ca²⁺ flashes, but significantly reduced Ca²⁺ wave frequency. Inhibition of mitochondrial Ca²⁺ uptake and Ca²⁺ release with Ru360 and CGP-37157, respectively, reduced the frequency of Ca²⁺ flashes and Ca²⁺ waves in GBSM. Similar to oligomycin, cyclosporin A did not alter AP and Ca²⁺ flash frequency but significantly reduced Ca²⁺ wave activity. These data suggest that mitochondrial Ca²⁺ handling is necessary for the generation and propagation of spontaneous electrical activity and may therefore play an important role in gallbladder tone and motility.