Our laboratory showed previously that estrogen activates ERK in neocortical cultures. To further elucidate the precise signaling sequelae that lead to estrogen-induced ERK activity, we evaluated the involvement of protein kinase C (PKC). We found that neocortical explants expressed primarily PKC gamma and PKC epsilon. Consistent with the involvement of PKC in mediating estrogen-induced ERK phosphorylation, we found that estrogen treatment induced translocation of these PKC isoforms to the plasma membrane. Importantly, inhibition of these isoforms abolished the ability of estrogen to phosphorylate ERK. While direct activation of PKC mimicked the effect of estrogen on ERK, both in pattern of activation and resulting intraneuronal distribution of ERK, PKC-induced ERK phosphorylation required the activity of MEK but not B-Raf. Collectively, these data suggest a critical role for PKC in mediating estrogen induction of ERK activation in the developing brain via a MEK-dependent but B-Raf-independent pathway.