Ca(2+)-dependent secretagogues (e.g., carbachol, histamine, ionomycin, and 4-bromo-A23187) have relatively transient effects on chloride secretion, even if there is a sustained increase in cytosolic calcium ([Ca2+]i) (as for the ionophores). Because these agents increase both [Ca2+]i and protein kinase C (PKC) activity, chloride secretion might be stimulated by [Ca2+]i and terminated by PKC activity. We tested the effect of a PKC activator, phorbol 12-myristate 13-acetate (PMA), on Cl- secretion by T84 cell monolayers by measuring short-circuit current (Isc). PMA alone had no effect on Isc but potentiated increases in Isc when added 10 min or less before Ca(2+)-dependent secretagogues. Chelation of [Ca2+]i with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid inhibited the increases both in [Ca2+]i and Isc induced by carbachol with or without brief PMA pretreatment. Longer preincubations with PMA inhibited Isc responses to Ca(2+)-dependent secretagogues, even when increased [Ca2+]i was sustained by ionophores. Inhibitors of PKC could reverse the inhibitory effect of PMA but did not reverse the potentiating effect. The effects of PMA on Cl- secretion were reproduced by 1,2-dioctanoyl-sn-glycerol and were mirrored by effects on K+ channel opening. Thus PMA has dual effects on chloride secretion. Initially, it exerts a stimulatory action and subsequently an inhibitory action. The stimulatory effect only occurs if Ca(2+)-dependent secretion is ongoing. The inhibitory effect of PMA is mediated by PKC and cannot be overcome by increasing [Ca2+]i.