Isolated parotid acinar cells embedded in Bio-Gel P-2 resin were perifused in small columns, and the effects of various agonists and their combinations on amylase release were studied. Isoproterenol gradually increased the rate of amylase secretion; its maximum response was attained about 5 min after the stimulation. A similar lime course of changes in amylase secretion was elicited by dibutyryl cyclic AMP, forskolin and isobutylmethylxanthine. Carbachol (CCh) and substance P evoked biphasic stimulation of amylase secretion; an initial rapid and large peak and a following sustained plateau. The magnitude of the maximum response induced by CCh or substance P was almost the same as that induced by isoproterenol. In Ca2+-free medium? CCh evoked only the initial peak and did not produce the sustained plateau, but the effect of isoproterenol was little changed. Amylase secretion induced by isoproterenol, but not by CCh and substance P, was markedly decreased by lowering the temperature of the medium. Combined addition of isoproterenol and 1 mu M CCh markedly augmented amylase secretion; the magnitude of its response was about three times that induced by isoproterenol alone. Potentiation was also observed between alpha- and beta-adrenergic receptors. Most of these results were very different from those obtained in botch systems. Thus, use of a perifusion system for analysis of amylase secretion is strongly recommended.