Tumor-promoting phorbol esters activate, but then deplete cells of, protein kinase C (PKC) with prolonged treatment. It is not known whether phorbol ester-induced tumor promotion is due to activation or depletion of PKC. In rat fibroblasts overexpressing the c-Src proto-oncogene, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced anchorage-independent growth and other transformation-related phenotypes. The appearance of transformed phenotypes induced by TPA in these cells correlated not with activation but rather with depletion of expressed PKC isoforms. Consistent with this observation, PKC inhibitors also induced transformed phenotypes in c-Src-overexpressing cells. Bryostatin 1, which inhibited the TPA-induced down-regulation of the PKCδ isoform specifically, blocked the tumor-promoting effects of TPA, implicating PKCδ as the target of the tumor-promoting phorbol esters. Consistent with this hypothesis, expression of a dominant negative PKCδ mutant in cells expressing c-Src caused transformation of these cells, and rottlerin, a protein kinase inhibitor with specificity for PKCδ, like TPA, caused transformation of c-Src-overexpressing cells. These data suggest that the tumor-promoting effect of phorbol esters is due to depletion of PKCδ, which has an apparent tumor suppressor function.