LH receptor activation leads to the phosphorylation/activation of p42/44 MAPK in preovulatory granulosa cells. As the LH receptor can activate both adenylyl cyclase and phospholipase C, we hypothesized that the LH receptor could elicit phosphorylation of p42/44 MAPK through activation of protein kinase A (PKA) and/or protein kinase C (PKC). Preovulatory granulosa cells in serum-free primary cultures were treated with ovulatory concentrations of human chorionic gonadotropin (hCG), an LH receptor agonist, with or without various inhibitors. The PKA inhibitor H89 as well as the myristoylated PKA inhibitor peptide PKI strongly inhibited hCG-stimulated p42/44 MAPK phosphorylation, whereas the PKC inhibitor GF109203X had no effect on p42/44 MAPK phosphorylation. LH receptor-stimulated phosphorylation of cAMP response element-binding protein (CREB), histone H3, and MAPK kinase (MEK) was also strongly inhibited by H89 and not by GF109203X. The extent of PKC activation was assessed in preovulatory granulosa cells using three criteria: translocation of PKC isoforms to the membrane fraction, phosphorylation of a known PKC substrate, and autophosphorylation of PKC delta on an activation-related site. By all three criteria PKCs were partially activated before hCG stimulation, and hCG treatment failed to elicit further PKC activation, in vitro or in vivo. Taken together, these results indicate that, under primary culture conditions where physiological levels of signaling proteins are present, hCG signals to activate MEK, p42/44 MAPK, CREB, and histone H3 in a predominantly PKA-dependent and PKC-independent manner. Unexpectedly, PKCs were partially activated in the absence of LH receptor activation, and LH receptor activation did not elicit further detectable PKC activation.