Transcriptional activation of the human glycoprotein hormone α-subunit (αGSU) promoter in response to GnRH and phorbol-12-myristate-13-acetate (PMA) has been well characterized in αT3-1 gonadotropes but not investigated in the more differentiated LβT2 clonal gonadotrope. We have evaluated αGSU transcription in the more mature LβT2 cell line, using deletion and heterologous constructs of the αGSU promoter linked to a luciferase reporter gene. Basal αGSU-promoter activity was significantly less in LβT2 cells than in αT3-1 cells, but stimulation of transfected cells with GnRH and PMA resulted in similar increases in αGSU-promoter activity. Deletional analysis of the human αGSU promoter in LβT2 cells indicated that sequences between −398 and −244 and between −244 and −195 base pairs (bp) were involved in regulating basal αGSU-promoter transcription, whereas the region between −244 and −195 bp regulated PMA-stimulated promoter activity. Deletion of this promoter region containing a steroidogenic factor-1 (SF-1) binding site abolished basal and PMA-stimulated transcription. Site-directed mutagenesis of the SF-1 binding site resulted in a significant attenuation of basal and PMA-stimulated αGSU transcription. Pretreatment of LβT2 cells with a mitogen-activated protein kinase kinase-specific inhibitor, U0126, abolished the PMA-stimulated increase in MAPK activity and significantly reduced basal and PMA-stimulated promoter activity. Electrophoretic mobility shift assays for SF-1 and GATA revealed that PMA failed to affect SF-1 binding but enhanced GATA binding to a consensus GATA oligonucleotide, an effect that was blocked with U0126 pretreatment, suggesting that GATA may mediate ERK activation of αGSU transcription. Our data suggests that, in the mature LβT2 gonadotrope cell line, two regions of the human αGSU promoter regulate basal transcription and that SF-1 is involved in mediating basal and PMA-stimulated promoter activity. Furthermore, PKC-stimulated transcription partially relies on ERK acting on elements downstream of −244 bp of the human αGSU promoter.