Phosphorylation of myristoylated alanine-rich protein kinase C substrate (MARCKS) in intact cells has been employed as an indicator for activation of protein kinase C (PKC). Specific PKC isoenzymes responsible for MARCKS phosphorylation under physiological conditions, however, remained to be identified. In our present study using stably transfected NIH 3T3 cell clones we demonstrate that expression of constitutively active mutants of either conventional cPKC-alpha or novel nPKC-epsilon increased phosphorylation of endogenous MARCKS in the absence of phorbol 12,13-dibutyrate in intact mouse fibroblasts, implicating that each of these PKC isoforms itself is sufficient to induce enhanced MARCKS phosphorylation. Similarly, ectopic expression of a constitutively active mutant of PKC-theta significantly increased MARCKS phosphorylation compared to vector controls, identifying PKC-theta as a MARCKS kinase. The PKC-specific inhibitor GF 109203X (bisindolylmaleimide I) reduced MARCKS phosphorylation in intact cells at a similar dose-response as enzymatic activity of recombinant isoenzymes cPKC-alpha, nPKC-epsilon, and nPKC-theta in vitro. Consistently, phorbol 12,13-dibutyrate-dependent MARCKS phosphorylation was significantly reduced in cell lines expressing dominant negative mutants of either PKC-alpha K368R or (dominant negative) PKC-epsilon K436R. The fact, that the constitutively active PKC-lambda A119E mutant did not alter the MARCKS phosphorylation underscores the assumption that atypical PKC isoforms are not involved in this process. We conclude that under physiological conditions, conventional cPKC-alpha and novel nPKC-epsilon, but not atypical aPKC-lambda are responsible for MARCKS phosphorylation in intact NIH 3T3 fibroblasts.