Recent studies have implicated protein kinase C (PKC) activation in drug resistance in vitro. PKC can be activated directly by phorbol-ester tumor promoters as well as by the bile acid deoxycholate. In this report, we demonstrate that deoxycholate, at concentrations that are chronically present in the lumen of the colon in vivo, mimicked phorbol-ester tumor promoters by protecting Adriamycin (ADR)-sensitive and multidrug-resistant (MDR) murine fibrosarcoma UV-2237M cells from ADR cytotoxicity. Deoxycholate also enhanced the resistance of the MDR cell line UV-2237M-ADRR to the cytotoxic effects of vincristine and vinblastine. In contrast to cytotoxic drug-selected MDR phenotypes, deoxycholate-induced drug resistance was transient and required continuous exposure to the bile acid. The protein kinase inhibitor H7 completely reversed the protection against ADR cytotoxicity conferred on UV-2237M-ADRR cells by deoxycholate, providing evidence that deoxycholate exerts its protective effects by a mechanism that involves stimulation of protein phosphorylation and not merely by detergent effects on membrane permeability. PKC consists of a family of at least seven isozymes with distinct modes of activation and substrate specificities. We previously reported that MDR UV-2237M cell lines contain higher levels of PKC activity than the parental ADR-sensitive UV-2237M cell line (O'Brian et al., FEBS Lett 246: 78-82, 1989). The present report shows that PKC-III is a major PKC isozyme in ADR-sensitive and MDR UV-2237M cell lines. Thus, the resistance to ADR induced by the phorbol esters in UV-2237M cell lines provides strong evidence that PKC-III activation confers protection against ADR on ADR-sensitive and MDR UV-2237M cell lines. Furthermore, since deoxycholate is an endogenous molecule in the colonic epithelium, our finding that physiological concentrations of deoxycholate can render cells more resistant to chemotherapeutic drugs in vitro may have implications for the biology and therapy of intestinal cancers.