Potentiation of amylase secretion by the combination of isoproterenol and substance P was examined in perfused rat parotid acinar cells. Combined additions of substance P and isoproterenol evoked biphasic changes in amylase secretion, an initial large peak and the following sustained plateau: the magnitudes of the both responses were higher than the sum of the responses induced by each agonist alone. Isoproterenol also increased the maximum response and the apparent affinity (EC50) for substance P to evoke the initial peak response; the EC50 values were about 20 and 0.8 nM, respectively, in the absence and the presence of isoproterenol. On the other hand, 1 nM substance P was sufficient for evoking the maximum potentiation of the sustained plateau response. Substance P did not change the EC50 for isoproterenol. The effect of isoproterenol was mimicked with dibutyryl cyclic AMP and agonists that increase parotid cyclic AMP. Omission of Ca2+ or addition of 5 mM nickel chloride almost completely abolished the potentiation of the sustained plateau, but little decreased that of the initial peak. Depletion of Ca2+ in InsP3-sensitive intracellular stores with thapsigargin, on the other hand, decreased the initial peak response, but not the sustained plateau, to substance P. The potentiation was also observed between isoproterenol and Ca2+ ionophores. Switching to the solutions containing higher concentrations of Ca2+ during the continuous stimulation with isoproterenol or IBMX evoked a large, but transient, response of amylase secretion. Time course of changes in amylase secretion induced by isoproterenol and substance P in combination was very similar to that of substance P, but not of isoproterenol. Isoproterenol did not enhance the effect of substance P on [Ca2+]i. These results show that the potentiation is mainly, if not totally, caused by cyclic AMP-induced enhancement of the potency and the efficacy in the pathway regulated by Ca2+.