The glycoprotein hormone alpha-subunit gene is expressed in a cell-specific manner in the anterior pituitary and placenta. Previous studies have shown that the region between -178 to -111 is indispensable for placental-specific expression of the human alpha-subunit gene. Using gene transfer techniques with chimeric luciferase plasmids, this report identifies regions of the mouse alpha-subunit promoter that are important for transcriptional activation in primary thyrotropic cells. Transient expression of a series of 5' flanking DNA deletions resulted in stepwise reductions of basal promoter activity between -480 to -417 (4-fold), -254 to -177 (5-fold), and -177 to -120 (3.5-fold). DNase-I protection analysis with nuclear extracts from thyrotropic tumor cells revealed specific protein-DNA interactions within each of these functionally defined regions. These were mapped to positions -474 to -452, -447 to -419, -213 to -170, and -158 to -101 within the 5' flanking region. In contrast, in mouse fibroblast L-cells no significant difference in alpha-subunit promoter activity was found by deleting the region from -480 to -177. However, a 3-fold decrease, similar to that found in primary thyrotropes, was found by deleting the region from -177 to -120. Further, a smaller region between -138 and -122 was the only area detected by the DNase-I protection assay using L-cell nuclear extracts. Thus, several cis-acting promoter elements located up-stream of position -177 are important for expression in thyrotropes. These elements also bind nuclear factors present in thyrotropes but not in nonpituitary fibroblasts and, therefore, differ from those mediating expression of the human alpha-subunit gene in the placenta.