Retinoic acid (RA) has been reported to inhibit the secretion and synthesis of the pituitary TSH in vivo and in vitro. However, little is known about the influence of RA on the expression of the prepro-TRH gene. We therefore investigated whether the promoter activity of the mouse TRH gene is directly regulated by RA using a transient transfection assay into CV-1 cells. In the absence of cotransfected RA receptor (RAR), all-trans-RA did not affect the promoter activity. In contrast, the cotransfected RARalpha significantly stimulated promoter activity in the absence of ligand, and all-trans-RA reversed basal promoter activation. The cotransfected thyroid hormone receptor-beta (TRbeta), but not 9-cis-RA receptor (RXR), had an additive effect on the RAR-dependent stimulation. TR and RAR can similarly interact with the corepressor proteins, and the cotransfected nuclear receptor corepressor (N-CoR) has been demonstrated to augment the transcriptional stimulation of the TRH gene by unliganded TR. As observed with TR, the coexpression of a N-CoR variant significantly enhanced the ligand-independent stimulation by RAR. A mutant RAR (RAR403) lacking the C-terminal activation function-2 (AF-2) activation domain that was essential for ligand-induced corepressor release constitutively stimulated the promoter activity. The constitutive stimulation by RAR403 was augmented by the cotransfected N-CoR variant. A deletion analysis of the 5'-flanking region of the TRH gene revealed that the minimal promoter region for the regulation by RAR was -83 to +53, with a consensus half-site motif for the thyroid hormone response element at -57. In contrast to the strong binding of TR to the thyroid hormone response element half-site in gel retardation assays, no binding of RAR homodimer, RAR/ RXR heterodimer, or RAR/TR heterodimer was observed to the minimal promoter region. These results collectively suggest that RAR without heterodimerization with RXR and TR regulates transcription of the mouse TRH gene in cooperation with the corepressor, and that the DNA binding of RAR appeared to be unnecessary for regulation of the TRH gene promoter.