We have previously shown that domains involved in binding of protein kinase C (PKC1) isozymes to their respective anchoring proteins (RACKs2) and short peptides derived from these domains are PKC isozyme-selective antagonists. We also identified PKC isozyme-selective agonists, named ψRACK3 peptides, derived from a sequence within each PKC with high homology to its respective RACK. We noted that all the ψRACK sequences within each PKC isozyme have at least one nonhomologous amino acid difference from their corresponding RACK that constitutes a charge change. Based on this information, we have devised here a new approach to design an isozyme-selective PKC antagonist, derived from the ψRACK sequence. We focused on εPKC ψRACK peptide, where the pseudo-εRACK sequence (ψεRACK; HDAPIGYD; corresponding to εPKC85-92) is different in charge from the homologous RACK-derived sequence (NNVALGYD; corresponding to εRACK285-292) in the second amino acid. Here we show that changing the charge of the ψεRACK peptide through a substitution of only one amino acid (aspartate to asparagine) resulted in a peptide with an opposite activity on the same cell function and a substitution for aspartate with an alanine resulted in an inactive peptide. These data support our hypothesis regarding the mechanism by which pseudo-RACK peptide activates PKC in heart cells and suggest that this approach is applicable to other signaling proteins with inducible protein-protein interactions.