We examined whether mitogen-activated protein (MAP) kinase was activated by stimulation of the cAMP pathway and whether MAP kinase activation was involved in synthesis of PRL and GH in GH(3) cells. Treatment of the cells with a cAMP analog, 8-(4-chlorophenylthio)cAMP (CPT-cAMP), activated MAP kinase and increased PRL at both the protein and messenger RNA levels. The protein and messenger RNA of GH were decreased by the treatment. We constructed the luciferase reporter genes after the promoters of PRL and GH and found the activation of both promoters by the CPT-cAMP treatment. We confirmed that overexpression of the catalytic subunit of cAMP-dependent protein kinase had essentially the same effects on MAP kinase activation and synthesis of PRL and GH as the CPT-cAMP treatment. Furthermore, treatment of the cells with pituitary adenylate cyclase-activating polypeptide 27 activated MAP kinase. The activation of PRL promoter by CPT-cAMP and pituitary adenylate cyclase-activating polypeptide 27 was abolished by pretreatment with PD098059 and H89. Although the increase in PRL and GH secretion by CPT-cAMP was inhibited by H89, PD098059 had no effect on secretion. These results suggest that cAMP-induced MAP kinase activation is essential for PRL gene expression, but not for secretion of PRL and GH.