Agents like carbachol and cholecystokinin (CCK), that activate chief cell phosphoinositidase C, thereby increasing cell calcium concentration, increase cAMP levels in cholera toxin-treated, but not control, gastric chief cells. In the present study, we found that phorbol esters, like PMA, that activate protein kinase C, also cause augmentation of chief cell cAMP levels. The maximal effect with PMA (100 nM) was about 50% of the maximal response with CCK (10 nM) or carbachol (100 microM). Because protein kinase C is a calcium-dependent enzyme, we examined the effect of modulating cell calcium levels with the ionophore A23187. The ionophore alone caused a dose-dependent augmentation of cAMP levels. Adding 100 nM PMA caused an additive response, such that a maximal cAMP response, equal to that seen with 100 microM carbachol, was observed with 30 nM A23187. Carbachol-, A23187-, and PMA-induced augmentation of cAMP levels was progressively reduced by increasing concentrations of calmidazolium, a calmodulin inhibitor. Combination of phorbol esters that activate protein kinase C with ionophores that increase cell calcium mimics the actions of CCK and carbachol on cAMP levels in cholera toxin-treated chief cells.