Protein kinase C (PKC) isozymes translocate to unique subcellular sites following activation. We previously suggested that translocation of activated isozymes is required for their function and that in addition to binding to lipids, translocation involves binding of the activated isozymes to specific anchoring proteins (receptors for activated protein kinase C. Using cultured cardiomyocytes we identified inhibitors, the V1 fragment of epsilonPKC (epsilonV1), and an 8-amino acid peptide derived from it that selectively inhibited the translocation of epsilonPKC. Inhibition of epsilonPKC translocation but not inhibition of delta or betaPKC translocation specifically blocked phorbol ester- or norepinephrine-mediated regulation of contraction. These isozyme-selective translocation inhibitors provide novel tools to determine the function of individual PKC isozymes in intact cells.