We examined the effects of sex steroids on prolactin promoter activity in rat somatolactotrophic GH3 cells. Both estradiol (E2) and progesterone (P4) were found to inhibit basal prolactin promoter activity, but to potentiate Thyrotropin-releasing hormone (TRH)-induced prolactin promoter activity. P4 had a greater inhibitory effect on basal prolactin promoter activity than E2, and P4 also potentiated TRH-induced prolactin promoter more potently than E2. Combined treatment with E2 and P4 further increased TRH-induced prolactin promoter activity. E2 and P4 also both reduced basal serum response element (SRE) promoter activity, and increased TRH-induced SRE promoter activity. Combination treatment with E2 and P4 reduced basal activity of SRE promoter and increased TRH-induced SRE activity more potently than E2 or P4 alone. In contrast, basal cAMP response element (CRE) promoter activity was not influenced by either E2 or P4, although TRH-induced CRE promoter was potentiated by each of these steroids, and was further increased by E2 and P4 combination treatment. Both E2 and P4 increased TRH-induced extracellular signal-regulated kinase (ERK) phosphorylation; however, intracellular cAMP levels was not influenced by E2 or P4. TRH-induced CRE promoter was inhibited by mitogen-activated protein kinase/ERK kinase (MEK) inhibitor and was increased by overexpression of MEK kinase (MEKK). This study showed that ERK and SRE transcriptional pathways, but not the cAMP/CRE pathway, may be involved in the suppression of basal prolactin promoter activity, whereas both the ERK/SRE and MAP kinase-mediated CRE pathways appear to be involved in the increased transcriptional efficiency of the prolactin promoter induced by TRH stimulation.