We have used a blot overlay assay to detect protein kinase C (PKC) interactions with other proteins. In many cases, the PKC binding proteins are also PKC substrates [Chapline et al. (1993) J. Biol. Chem. 268, 6858]. The purpose of the current studies was to characterize the PKC domains involved in the interactions with other proteins. alpha, beta, and epsilon isoforms of PKC interact with the same binding proteins in fibroblast cell extracts. These results indicate that constant rather than isozyme-specific (variable) regions are the major determinants of the interactions studied. PKC binding required phosphatidylserine (PS), indicating that the PS binding regulatory domain of PKC is involved in the interactions. The PKC pseudosubstrate peptide sequence, which is contained within the regulatory domain, also showed PS-dependent binding to the PKC binding proteins. To further investigate the role of the pseudosubstrate peptide in promoting PKC-protein interactions, an N-terminal truncation mutant lacking the pseudosubstrate sequence was prepared. Binding of the mutant alpha-PKC was diminished compared to wild-type alpha-PKC, although some binding was still apparent. These results indicate that the pseudosubstrate sequence contributes to, but is not the sole determinant of, PKC binding activity.