To elucidate the role of protein kinase C (PKC) in nerve growth factor (NGF)-induced differentiation, PMA downregulation of pheochromocytoma (PC12) cells was undertaken. Prolonged treatment (2 d) of PC12 cells with PMA (1 microM) resulted in depleting the cells of alpha, beta, delta, and epsilon-PKC isoforms, but had no effect on the expression of the atypical PKC isoform zeta. PC12 cells, which expressed only PKC zeta, were evaluated for their responses to NGF. Removal of the PMA-sensitive PKC isoforms enhanced the ability of NGF to promote neurite extension. Both the percentage cells with neurites and length of neurites were increased in the PMA-treated cells, whereas no effect was observed on the number of neurites per cell or branching of individual neurites. In addition, PMA downregulation resulted in an increase in the incorporation of 3H-thymidine without any significant effect on the expression of c-fos. Addition of NGF to PC12 cells depleted of the PMA-sensitive PKC isoforms resulted in the activation of PKC zeta (Wooten et al., 1994). To test whether the transient activation of PKC zeta is a necessary component of the neuritogenetic pathway, antisense oligonucleotide strategy was utilized to remove this particular PKC isoform. The addition of a 20-bp antisense oligonucleotide directed against the 5' coding sequence of PKC zeta attenuated NGF-induced neurite outgrowth in PC12 cells lacking PMA-sensitive PKC isoforms. Sense oligonucleotide directed at the same site was without effect on NGF responses. These data indicate that PKC zeta comprises a portion of the NGF pathway and underscores the importance of this isoform in neuronal differentiation. Moreover, these findings demonstrate that the PMA-insensitive pathway, which was previously characterized as PKC-independent, and the neurite induction pathway are synonymous and mediated by PKC zeta.