Although activation of protein kinase C (PKC) &egr; and mitogen-activated protein kinases (MAPKs) are known to play crucial roles in the manifestation of cardioprotection, the spatial organization of PKC&egr; signaling modules in naïve and protected myocardium remains unknown. Based on evidence that mitochondria are key mediators of the cardioprotective signal, we hypothesized that PKC&egr; and MAPKs interact, and that they form functional signaling modules in mitochondria during cardioprotection. Both immunoblotting and immunofluorescent staining demonstrated that PKC&egr;, ERKs, JNKs, and p38 MAPK co-localized with cardiac mitochondria. Moreover, transgenic activation of PKC&egr; greatly increased mitochondrial PKC&egr; expression and activity, which was concomitant with increased mitochondrial interaction of PKC&egr; with ERKs, JNKs, and p38 as determined by co-immunoprecipitation. These complex formations appeared to be independent of PKC&egr; activity, as the interactions were also observed in mice expressing inactive PKC&egr;. However, although both active and inactive PKC&egr; bound to all three MAPKs, increased phosphorylation of mitochondrial ERKs was only observed in mice expressing active PKC&egr; but not in mice expressing inactive PKC&egr;. Examination of potential downstream targets of mitochondrial PKC&egr;-ERK signaling modules revealed that phosphorylation of the pro-apoptotic protein Bad was elevated in mitochondria. Together, these data show that PKC&egr; forms subcellular-targeted signaling modules with ERKs, leading to the activation of mitochondrial ERKs. Furthermore, formation of mitochondrial PKC&egr;-ERK modules appears to play a role in PKC&egr;-mediated cardioprotection, in part by the phosphorylation and inactivation of Bad.