Protein kinase C (PKC) &egr; and PKC&dgr; translocation in neonatal rat ventricular myocytes (NRVMs) is accompanied by subsequent activation of the ERK, JNK, and p38MAPKcascades; however, it is not known if either or both novel PKCs are necessary for their downstream activation. Use of PKC inhibitors to answer this question is complicated by a lack of isoenzyme specificity, and the fact that many PKC inhibitors stimulate JNK and p38MAPKactivity. Therefore, replication-defective adenoviruses (Advs) encoding constitutively active (ca) mutants of PKC&egr; and PKC&dgr; were used to test if either or both of these PKCs are sufficient to activate ERKs, JNKs, and/or p38MAPKin NRVMs. Adv-caPKC&egr; infection (1 to 25 multiplicities of viral infection (MOI); 4 to 48 hours) increased total PKC&egr; levels in a time- and dose-dependent manner, with maximal expression observed 8 hours after Adv infection. Adv-caPKC&egr; induced a time- and dose-dependent increase in phosphorylated p42 and p44 ERKs, as compared with a control Adv encoding &bgr;-galactosidase (Adv-ne&bgr;gal). Maximal ERK phosphorylation occurred 8 hours after Adv infection. In contrast, JNK was only minimally activated, and p38MAPKwas relatively unaffected. Adv-caPKC&dgr; infection (1 to 25 MOI, 4 to 48 hours) increased total PKC&dgr; levels in a similar fashion. Adv-caPKC&dgr; (5 MOI) induced a 29-fold increase in phosphorylated p54 JNK, and a 15-fold increase in phosphorylated p38MAPK24 hours after Adv infection. In contrast, p42 and p44 ERK were only minimally activated. Whereas neither Adv induced NRVM hypertrophy, Adv-caPKC&dgr;, but not Adv-caPKC&egr;, induced NRVM apoptosis. We conclude that the novel PKCs differentially regulate MAPK cascades and apoptosis in an isoenzyme-specific and time-dependent manner.