Thyrotropin (TSH), a pituitary glycoprotein, consists of two subunits (alpha and beta), and regulates thyroid hormone production by the thyroid gland. The thyroid hormones, in turn, feedback negatively on TSH production. We have investigated the effect of triiodothyronine (T3) on the transcription of the TSH subunit genes. Hypothyroid LAF1 mice bearing TtT 97 thyrotropic tumors were injected daily with T3 (5 micrograms/100 g of body weight, intraperitoneal) and killed after 0, 0.5, 1, 4, or 120 h. Nuclei were isolated from the tumors and allowed to continue RNA synthesis in the presence of [alpha-32P]UTP. Newly synthesized mRNA sequences were quantitated by hybridization to immobilized cloned cDNA containing sequences specific for either alpha subunit or TSH-beta mRNA. Basal levels of mRNA synthesis were similar for alpha subunit and TSH-beta. After only 30 min of T3 treatment in vivo, mRNA synthesis was decreased for both alpha subunit (by 28%) and TSH-beta (by 61%). These decreases were maximum after 4 h, (75% for alpha subunit, greater than 95% for TSH-beta mRNA synthesis), and were maintained for the 5 days of T3 treatment. Decreases in mRNA synthesis precede decreases in tissue mRNA concentrations. Our studies suggest that T3 rapidly decreases the transcription of both the alpha subunit and TSH-beta genes, and that the transcription of the TSH-beta gene is affected to a greater extent than the alpha subunit gene.