Steroidogenic factor-1 (SF-1) is a key regulator of endocrine development, and mediates expression of gonadotrophin-specific genes in the pituitary. Basal and hormone stimulated transcription of the human glycoprotein hormone alpha-subunit gene (alphaGSU) in gonadotrophs involves SF-1 and its cognate binding site, the gonadotroph-specific element (GSE). In this study, we demonstrate that SF-1 significantly enhances basal and forskolin-stimulated transcription of the human alphaGSU promoter in GH(3) cells. Mutation of the GSE abolished the SF-1-mediated transactivation of basal alphaGSU promoter activity, and significantly attenuated the forskolin effect by 50%. Mutation of the Ser203 residue in SF-1 to Ala blocked basal transactivation of alphaGSU promoter activity, and halved the forskolin effect. These data collectively reveal a direct role for SF-1 and the GSE in mediating basal and forskolin-stimulated transcription of the human alphaGSU promoter in GH(3) cells. The phosphorylation site at Ser203 appears to be required for these effects.