Receptors for activated C kinase (RACKs) have been shown to facilitate activation of protein kinase C (PKC). However, it is unknown whether PKC activation modulates RACK protein expression and PKC-RACK interactions. This issue was studied in two PKCε transgenic lines exhibiting dichotomous cardiac phenotypes: one exhibits increased resistance to myocardial ischemia (cardioprotected phenotype) induced by a modest increase in PKCε activity (228 ± 23% of control), whereas the other exhibits cardiac hypertrophy and failure (hypertrophied phenotype) induced by a marked increase in PKCε activity (452 ± 28% of control). Our data demonstrate that activation of PKC modulates the expression of RACK isotypes and PKC-RACK interactions in a PKCε activity- and dosage-dependent fashion. We found that, in mice displaying the cardioprotected phenotype, activation of PKCε enhanced RACK2 expression (178 ± 13% of control) and particulate PKCε-RACK2 protein-protein interactions (178 ± 18% of control). In contrast, in mice displaying the hypertrophied phenotype, there was not only an increase in RACK2 expression (330 ± 33% of control) and particulate PKCε-RACK2 interactions (154 ± 14% of control) but also in RACK1 protein expression (174 ± 10% of control). Most notably, PKCε-RACK1 interactions were identified in this line. With the use of transgenic mice expressing a dominant negative PKCε, we found that the changes in RACK expression as well as the attending cardiac phenotypes were dependent on PKCε activity. Our observations demonstrate that RACK expression is dynamically regulated by PKCε and suggest that differential patterns of PKCε-RACK interactions may be important determinants of PKCε-dependent cardiac phenotypes.