Benzoyl peroxide (BP), used widely in dermatologic therapy and by the food industry, is considered a tumor promoter in chemically induced skin. Tumor promoters of both the phorbol and non-phorbol type interact with protein kinase C (PKC). This enzyme, therefore, is regarded as the intracellular receptor for a number of tumor promoters. BP bears some structural resemblance to diacylglycerol (DAG) and thus may exert its action through the PKC system. Based on these observations, we have investigated the effect of BP on PKC from mouse skin. Our data show that unlike phorbol esters, which stimulate PKC (in vivo and in vitro), BP inhibits PKC. Concentration-dependent inhibition by BP is observed when PKC is stimulated by phorbol esters, diacylglycerol, phosphatidyl serine (PS), or a combination of the latter two. BP also inhibits PKC stimulated by (-) Indolactam V, a nonphorbol compound resembling the teleocidins. 3H-phorbol ester binding experiments reveal that inhibition by BP may be due to its interference with the phorbol ester binding site and consequently diacylglycerol binding. The binding data and the inability of BP to inhibit either cyclic AMP-dependent protein kinase I or II imply that BP interacts with PKC, and not with the histone substrate. Results presented here clearly indicate that unlike phorbol and certain non-phorbol type of tumor promoters BP does not stimulate PKC in vitro.