The action of propionate, one of the major end products of microbial fermentation in herbivores was investigated in isolated, perifused pancreatic acini of guinea pigs, voles, and mice. With the use of guinea pig acini, 100 µM propionate had no effect, whereas 300 and 600 µM increased amylase release by six- and ninefold, respectively. Simultaneous perifusion of carbachol (CCh) 10 µM plus propionate 100 µM in guinea pig acini produced a potentiated secretory response that was 130% higher than the summated value obtained with CCh and propionate alone. The potentiation by propionate (100 µM) of CCh (10 µM)-induced amylase release was also obtained in vole pancreatic acini, but the mouse pancreatic preparation did not exhibit a similar potentiation. In contrast to CCh, propionate (100-600 µM) alone had no significant effect on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and did not alter [Ca²⁺]_i elicited by CCh. Ca ionophore A23187 (5 µM)-induced amylase release in guinea pig acini was enhanced twofold by the addition of propionate. Cellular cAMP content was increased slightly by propionate, but did not alter dose dependently. The cAMP level with combinations of CCh and propionate was almost same as that with CCh alone and propionate alone. Staurosporine did not modify amylase secretion induced by a combination of CCh and propionate. These results suggest that propionate, in addition to a direct action on amylase release, potentiates CCh-induced amylase release in guinea pig and vole acini via a secretory pathway not associated with an increase in [Ca²⁺]_i and cellular cAMP.