The effects of physiological concentrations of L-T3 (T3) were examined in dispersed cell cultures of pituitaries obtained from 10- to 12-day-old rats. T3 inhibited TSH secretion by 50% and blunted the TSH response to TRH. The PRL response to TRH was also inhibited by T3, and GH secretion was increased 2-fold. These responses were half-maximal at 0.1 nM added T3 in medium supplemented with 10% hypothyroid calf serum, corresponding to a free T3 concentration of 5 pM. In the presence or absence of added T3, TRH effects were half-maximal at 0.5-3 nM, and T3 suppression was not overcome by high concentrations of TRH (up to 1 microM). Maximal inhibition of TSH responses to TRH occurred when cultures were preincubated with thyroid hormone for 24 h; a significant effect was observed after 8 h. The specific binding of [3H]TRH to dispersed rat pituitary cells was decreased 55-70% by T3 in a dose-dependent manner. Inhibition of TSH secretion by T3 was reversible within 24 h, and the fraction of thyrotrophs in the cultures (0.22) was not altered by T3 over the course of the experiments. The results demonstrate that physiological concentrations of T3 regulate TSH and PRL responses to TRH and control TRH receptor levels by a direct action on normal rat pituitary cells.