We have previously demonstrated that low concentrations of phorbol esters stimulate the selective translocation of protein kinase C (PKC) alpha and epsilon from the cell soluble to the particulate fraction in NCMs (neonatal rat cardiac myocytes). We therefore determined if the in vitro phosphorylation of substrates in these fractions could be used as assays of PKCalpha or epsilon activation. Intact cell phorbol ester treatment caused a decline in the in vitro (32)P-incorporation into several proteins in the cell-soluble fraction. These declines occurred in the presence or absence of in vitro Ca(2+) and probably reflected the exit of PKC isoenzymes from the soluble fraction. In contrast, an approx. 18 kDa protein incorporated (32)P in particulate fractions isolated from 4beta-PMA-treated cells in a Ca(2+)-independent manner. Proteomic and immunoprecipitation analyses indicated that the protein is subunit IV of the cytochrome c oxidase complex (COIV). In vitro phosphorylation of COIV was attenuated by PKC pseudosubstrate peptides. Introduction of an PKCepsilon-selective translocation inhibitor [Johnson, Gray, Chen and Mochly-Rosen (1996) J. Biol. Chem. 271, 24962-24966] into NCMs before 4beta-PMA treatments also attenuated the in vitro phosphorylation of COIV. In mitochondrial extracts from 4beta-PMA-treated NCMs, the PKCepsilon isoenzyme coimmunoprecipitated with COIV, and cytochrome c oxidase activity was enhanced 2-fold. The in vitro phosphorylation of COIV reflects a novel approach for monitoring PKCepsilon function in NCMs. Furthermore, PKCepsilon probably interacts with COIV in NCM mitochondria to enhance electron-transport chain complex IV activity.