Pituitary expression of the glycoprotein hormone alpha-subunit gene localizes to thyrotropes and gonadotropes. Factors that activate the alpha-subunit promoter exclusively in thyrotropes have not been described. In the current studies, alpha-subunit promoter activity was compared in alphaTSH thyrotropic and alphaT3 gonadotropic cells. Deletional analysis revealed a more important contribution of the -453/-381 region in thyrotropic cells and the -341/-297 region in gonadotropic cells, while other deletions had similar effects. Mutational analysis revealed some regions that were functionally similar, while others were active only in thyrotropic cells, such as the regions - 434/ - 421, - 398/ - 385, - 376/- 364 and - 363/- 351, that decreased activity by approximately 2-fold each. Combined mutation of the regions - 434/ - 421, - 398/ - 385 and - 376/ - 364 decreased activity by 5-fold in thyrotropic cells and not at all in gonadotropic cells. Cotransfection with Ptx1 in CV-1 cells resulted in a strong stimulation of alpha-subunit promoter activity, that was significantly diminished with mutation of the - 398/ - 385 region that disrupts the Ptx1 binding site, suggesting that this factor may play a role in thyrotrope-specific activation. This analysis provides basic information important to identify common and unique factors contributing to the cell-specific expression of the alpha-subunit gene.