Maladaptive cardiac hypertrophy results in phenotypic changes in several genes that are thyroid hormone-responsive suggesting that thyroid hormone receptor (TR) function may be altered by cellular kinases, including protein kinase C (PKC) isozymes that are activated in pathologic hypertrophy. To investigate the role of PKC signaling in regulating TR function, cultured neonatal rat ventricular myocytes were transduced with adenovirus-expressing wild type (wt) or kinase inactive (dn) PKC, or constitutively active (ca) PKC and PKC. Overexpression of wtPKC, but not caPKC or caPKC, induced a 28-fold increase (p<0.001) in TR1 protein in the nuclear compartment and a smaller increase in the cytosol. Furthermore, TR1 mRNA was increased 55-fold (p<0.001). This effect of PKC was dependent on its kinase activity since dnPKC was without effect. Phorbol myristate acetate (PMA) induced nuclear translocation of endogenous PKC and Ad-wtPKC, concomitantly with an increase in nuclear TR1 protein. In contrast, PMA-induced nuclear translocation of dnPKC resulted in a decrease of TR1. The increase in TR1 protein in Ad-wtPKC-transduced cardiomyocytes was not the result of a reduced rate of protein degradation; nor was the half-life of TR1 mRNA prolonged, suggesting a PKC-mediated effect on TR1 transcription. Although phosphorylation of ERK1/2 was increased in Ad-wtPKC-transduced cells, inhibition of pERK did not change TR1 expression. PKC overexpression in cardiomyocytes caused marked repression of T3-responsive genes, -myosin heavy chain and SERCA2. Treatment with T3 for 4 hrs resulted in significant reductions of PKC in nuclear and cytosolic compartments, and decreased TR1 mRNA and protein, with normalization of phenotype. These results implicate PKC as a regulator of TR function and suggest that nuclear localization of PKC may control transcription of the TR gene, and consequently, affect cardiac phenotype.