In this study, we investigated the effects of secretagogues and bile acids on the mitochondrial membrane potential of pancreatic acinar cells. We measured the mitochondrial membrane potential using the tetramethylrhodamine-based probes tetramethylrhodamine ethyl ester and tetramethylrhodamine methyl ester. At low levels of loading, these indicators appeared to have a low sensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and no response was observed to even high doses of cholecystokinin. When loaded at high concentrations, tetramethylrhodamine methyl ester and tetramethylrhodamine ethyl ester undergo quenching and can be dequenched by mitochondrial depolarization. We found the dequench mode to be 2 orders of magnitude more sensitive than the low concentration mode. Using the dequench mode, we resolved mitochondrial depolarizations produced by supramaximal and by physiological concentrations of cholecystokinin. Other calcium-releasing agonists, acetylcholine, JMV-180, and bombesin, also produced mitochondrial depolarization. Secretin, which employs the cAMP pathway, had no effect on the mitochondrial potential; dibutyryl cAMP was also ineffective. The cholecystokinin-induced mitochondrial depolarizations were abolished by buffering cytosolic calcium. A non-agonist-dependent calcium elevation induced by thapsigargin depolarized the mitochondria. These experiments suggest that a cytosolic calcium concentration rise is sufficient for mitochondrial depolarization and that the depolarizing effect of cholecystokinin is mediated by a cytosolic calcium rise. Bile acids are considered possible triggers of acute pancreatitis. The bile acids taurolithocholic acid 3-sulfate, taurodeoxycholic acid, and taurochenodeoxycholic acid, at low submillimolar concentrations, induced mitochondrial depolarization, resolved by the dequench mode. Our experiments demonstrate that physiological concentrations of secretagogues and pathologically relevant concentrations of bile acids trigger mitochondrial depolarization in pancreatic acinar cells.