Protein kinase C (PKC) isoenzymes play a critical role in cardiomyocyte hypertrophy. At least 3 different phorbol ester-sensitve PKC isoenzymes are expressed in neonatal rat ventricular myocytes (NRVM): PKC, PKC and PKC. Using replication-defective adenoviruses (Adv) expressing wild-type (wt) and dominant-negative (dn) PKC together with phorbol myristate acetate (PMA), a hypertrophic agonist and activator of all 3 PKC isoenzymes, we studied the role of PKC in signaling specific aspects of the hypertrophic phenotype. PMA induced nuclear translocation of endogenous and Adv-wtPKC in NRVM. wtPKC overexpression increased protein synthesis (PS) and protein/DNA ratio (P/D) but did not affect cell surface area (CSA) or cell shape as compared to uninfected or control Adv -galactosidase (Adv-gal) infected cells. PMA-treated, uninfected cells displayed increased PS, P/D and CSA, and elongated morphology. PMA did not further enhance PS or P/D in Adv-wtPKC infected cells. To assess the requirement of PKC for these PMA-induced changes, Adv-dnPKC or Adv-gal infected NRVM were stimulated with PMA. Without PMA, Adv-dnPKC had no effect on PS, P/D, CSA, or shape, versus Adv-gal. PMA increased PS, P/D and CSA in Adv-gal infected cells, but these parameters were significantly reduced in PMA-stimulated, Adv-dnPKC infected NRVM. Overexpression of dnPKC enhanced PMA-induced cell elongation. Neither wtPKC nor dnPKC affected ANF gene expression. Insulin like growth factor-1 (IGF-1) also induced nuclear translocation of endogenous PKC. PMA induced ERK1/2 activation but wtPKC overexpression did not. However, Adv-dnPKC partially blocked PMA-induced ERK activation. Thus PKC is necessary for certain aspects of PMA-induced NRVM hypertrophy.