TSH is expressed in two populations of thyrotropes in the pituitary: one in the pars distalis and a second in the pars tuberalis. Pars distalis thyrotropes exhibit classical endocrine inhibition of TSH by thyroid hormone, whereas pars tuberalis thyrotropes do not. The majority of our understanding of TSH subunit gene regulation has come from studies conducted in dispersed pituitary, dispersed thyrotropic tumors, or the GH3 somatolactotrope cell line. However, the dispersed pituitary model is limited because of its inherent heterogeneity, thyrotropic tumors are difficult to grow and maintain, and the GH3 cells lack endogenous TSH expression. The recent derivation of a clonal thyrotrope cell line, T alphaT1, that expresses thyrotrope-specific markers, overcomes these limitations. However, because it was not possible to distinguish whether the tumor from which the T alphaT1 cells are derived originated in the pars distalis or the pars tuberalis, it was necessary to define their cellular origin and thereby establish their status as representative thyrotrope cells for future molecular studies. In this study, we demonstrate that the T alphaT1 cells express thyroid hormone receptors (beta1 and beta2) and their heterodimeric partner, retinoid X receptor-gamma. Treatment with T3 causes a dose- and time-dependent decrease in the expression of the TSH beta-subunit messenger RNA. In contrast to previous reports in rat pituitary cultures, T3 does not alter TSH beta-subunit messenger RNA stability in the T alphaT1 cells. Based on these data and the presence of thyrotrope-specific isoforms of the transcription factor Pit-1, we conclude that the T alphaT1 cells represent differentiated thyrotropes of the pars distalis and will be a useful model system for future analysis of the cis- and trans-acting factors necessary for thyrotrope-specific and thyroid hormone-regulated TSH gene expression.