Cardiac fibroblasts (CFs) are the major non-muscle cell type of the myocardium, and the primary role of this cell type is to regulate the extracellular matrix of the heart. Hyperactivation of CFs occurs following myocardial infarction and during chronic hypertension, leading to significant fibrosis of the myocardium and a detrimental loss of contractility. Angiotensin II (ANG II) is a major effector of CF activation and its production is increased during these cardiovascular pathologies. A critical initial step in CF activation is cellular proliferation. ANG II induces CF proliferation through activation of the ERK 1/2 cascade, but the mechanism by which ANG II activates ERK in CFs remains unclear. The goal of the current study is to determine whether PKCζ, c-src, PI3-K or the EGF receptor mediate ANG II-induced ERK activation in adult rat CFs. We found that inhibition of PKCζ using a myristolated psuedosubstrate significantly reduced the extent of ERK 1/2 activation by ANG II. Additionally, the PKCζ pseudosubstrate reduced ANG II-induced [3H]-thymidine incorporation in CFs. However, selective inhibition of c-src, PI3-K, and the EGF receptor neither affected the levels of phosphorylated ERK 1/2, nor reduced ANG II-induced [3H]-thymidine incorporation into CFs. This study therefore identifies PKCζ as an important downstream mediator of ANG II-induced ERK 1/2 activation and proliferation in cardiac fibroblasts. This study is supported by Ohio Board of Regents Grant #34177