Protein kinase C (PKC) has been shown to regulate the ethanol sensitivity of membrane-bound receptors and transporters, but little is known about the molecular mechanisms underlying this regulation. PKC is a family of isozymes that translocate to new intracellular sites on activation. Here we present immunochemical data showing that ethanol causes translocation of δ- and ε-PKC to new intracellular sites. Ethanol causes translocation of δ-PKC from the Golgi to the perinucleus; this translocation is similar to that induced by activation of PKC with phorbol esters. In contrast, ε-PKC translocation caused by ethanol is different from that induced by phorbol esters; ethanol causes translocation of ε-PKC from the perinucleus to the cytoplasm, whereas phorbol ester activation causes translocation of ε-PKC to the nucleus. Because the substrate specificity of these kinases is determined by their site of localization, ethanol-induced translocation of δ- and ε-PKC to new intracellular sites may explain some of the pleiotropic effects of ethanol on cellular functions.