Regulation of intracellular free calcium ([C a2 +]i) in single epithelial duct cells of isolated rat and guinea pig pancreatic interlobular ducts by secretin, carbachol and cholecystokinin was studied by microspectrofluorometry using the Ca 2 +-sensitive, fluorescent probe Fura-2. Rat and guinea pig duct cells exhibited mean resting [Ca2+]i of 84 nM and 61 nM, respectively, which increased by 50% -100% in response to carbachol stimulation, thus demonstrating the presence of physiologically responsive cholinergic receptors in pancreatic ducts of both species. The carbachol-induced increase in [Ca2+]i involved both mobilization of Ca 2+ from intracellular stores and stimulation of influx of extracellular Ca z +. In contrast, neither cholecystokinin nor secretin showed reproducible or sizeable increses in [Ca z+]i. Both rat and guinea pig duct cells showed considerable resting Ca 2 + permeability. Lowering or raising the extracellular [Ca2+]i led, respectively, to a decrease or increase in the resting [Ca2+]i. Application of Mn 2+ resulted in a quenching of the fluorescence signal indicating its entry into the cell. The resting Ca 2+ and Mn 2+ permeability could be blocked by La 3 + suggesting that it is mediated by a Ca 2 + channel.