We examined the role of PKCs and Ca2+ in GnRH-stimulated p38MAPK phosphorylation in the gonadotrope derived alphaT3-1 and LbetaT2 cell lines. GnRH induced a slow and rapid increase in p38MAPK phosphorylation in alphaT3-1 and LbetaT2 cells respectively, while PMA gave a slow response. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs), has revealed differential role for PKCalpha, PKCbetaII, PKCdelta and PKCepsilon in p38MAPK phosphorylation in a ligand-and cell context-dependent manner. The paradoxical findings that PKCs activated by GnRH and PMA play a differential role in p38MAPK phosphorylation may be explained by differential localization of the PKCs. Basal, GnRH- and PMA- stimulation of p38MAPK phosphorylation in alphaT3-1 cells is mediated by Ca2+ influx via voltage-gated Ca2+ channels and Ca2+ mobilization, while in the differentiated LbetaT2 gonadotrope cells it is mediated only by Ca2+ mobilization. p38MAPK resides in the cell membrane and is relocated to the nucleus by GnRH ( approximately 5 min). Thus, we have identified the PKCs and the Ca2+ pools involved in GnRH stimulated p38MAPK phosphorylation.