Stimulation of secretion by muscarinic agonists in guinea pig parotid or pancreatic acini is accompanied by a translocation of protein kinase C (PKC) from the cytosol to the particulate fraction [Machado-De Domenech and Söling (1987) Biochem. J. 242, 749-754] and by a PKC-mediated phosphorylation of the ribosomal protein S6 [Padel and Söling (1985) Eur. J. Biochem. 151, 1-10]. In order to decide whether PKC is directly involved in the secretory process, the effect of down regulation of PKC by phorbol 12-myristate 13-acetate (PMA) was studied in primary cultured guinea pig parotid acinar cells. These cells secrete in response to carbachol and isoproterenol. Only the carbachol response is associated with an increase in cytosolic calcium. Carbachol plus isoproterenol lead to an over-additive stimulation of secretion, an effect which depends completely on the presence of external calcium. Down regulation of PKC by about 90% did not significantly affect carbachol-induced exocytosis, whereas isoproterenol-stimulated secretion was almost doubled. The secretory response to permeable cAMP analogues was also enhanced in PKC-down-regulated acini, indicating a post-receptor effect. The increased response to isoproterenol was also observed in the absence of external calcium. The isoproterenol effect was significantly inhibited by the relatively specific cAMP-dependent protein kinase inhibitor H-89, which had only a minor effect on carbachol-induced exocytosis. Although down regulation of total PKC by up to 90% did not significantly affect the secretory response to carbachol, RO 31-8220, a relatively specific inhibitor of PKC, abolished carbachol-induced secretion in normal as well as in PMA-down-regulated cells. This indicates that a PKC isoform resistant to down regulation by PMA is involved in carbachol- but not in cAMP-mediated secretion.