We investigated the effects of the protein kinase C (PKC¹) stimulating 12-deoxyphorbol 13-phenylacetate 20-acetate (DOPPA) and phorbol 12-myristate 13-acetate (PMA) phorbol esters on cAMP-dependent, forskolin (FSK)-stimulated, short-circuited chloride current (I_sc-cAMP) generation by colonocyte monolayers. These agonists elicited different actions depending upon dose and incubation time; acute PMA effects (< 5 min) were independent of cAMP-agonist and were characterized by transient anion-dependent transcellular (Tc) and apical membrane (Ap) I_sc generation. DOPPA failed to elicit similar responses. While chronic (24 hr) exposure to both agents inhibited FSK-stimulated Tc and Ap I_sc-cAMP, the effects of DOPPA were more complex: This conventional PKC-specific agonist also stimulated barium-sensitive basolateral (Bl) membrane dependent facilitation of Tc I_sc-cAMP. PMA did not elicit a similar phenomenon. Prolonged exposure to high dose PMA but not DOPPA led to Ap Isc-cAMP recovery. Changes in PKC , 1, and isoform membrane partitioning and expression correlated with these findings. PMA induced Tc I_sc correlated with PKC membrane association whereas, low doses of both agents inhibited Tc and Ap Isc-cAMP, increased PKC1 and decreased PKC2 membrane association and caused reciprocal changes in isoform mass. During Ap I_sc-cAMP recovery after prolonged high dose PMA exposure, almost complete depletion of cellular cPKC1 and a significant reduction in PKC isoform mass occurred. Thus, activated PKC 1 and/or PKC prevented, whereas activated PKC facilitated, Ap I_sc-cAMP. PKC-dependent augmentation of Tc I_sc-cAMP at the level of the basolateral membrane demonstrated that transport events with geographically distinct subcellular membranes can be independently regulated by the PKC isoform.