This study was designed to construct the primary culture system to detect the change in TSH beta subunit (TSH beta) gene expression in individual cells. Adult, male Wistar rats were sacrificed by transcardial perfusion of 0.25% trypsin solution under pentobarbital anesthesia (50 mg/kg body weight). Their anterior pituitaries were removed, dispersed and cultured for 1, 2, 3, or 6 days with or without 1 nM triiodothyronine (T3) under the serum-free condition. In some cultures, TRH was added to a final concentration of 1 microM on 6, 12 or 24 h before fixation. Then the culture media were removed to measure TSH concentration. Cells were fixed with paraformaldehyde and hybridized with 35S-labeled RNA probe complementary to TSH beta mRNA. Emulsion autoradiography was subsequently performed. T3 treatment markedly suppressed relative cellular levels of TSH beta mRNA on 2, 3 and 6 days after the onset of culture (day 2, 3 and 6) and suppressed TSH secretion on day 3 and 6. TRH treatment increased TSH beta mRNA on 12 and 24 h after the treatment on day 2 and 3 but did not increase TSH beta mRNA on day 6. TSH concentration in the culture medium was increased by TRH treatment on 6, 12 and 24 h after the treatment on day 2, on 12 h and 24 h on day 3, and 24 h on day 6. On day 2 and 3, although T3 treatment suppressed basal level of TSH beta mRNA, TRH-induced increase in TSH beta mRNA was not suppressed by T3 treatment. These results show that the thyroid hormone and TRH regulate TSH beta gene expression independently. Our culture system may provide a useful model to examine the action of individual substances on a specific subpopulation of the anterior pituitary cells.