The addition of thyroid hormone to cultures of GH3 or GH4C1 pituitary tumor cells maintained in medium with hypothyroid serum decreased the concentration of specific receptors for TRH. The relationship between thyroid hormone effects on TRH receptors and TRH responses was examined by testing the concentration dependence, time course, and specificity of these changes. The concentrations of T3 giving half-maximal decreases in [3H]TRH binding and inhibition of the PRL response to TRH were 0.20 and 0.24 nM, respectively. TRH stimulated the rate of [3H]uridine uptake by 50% in cultures incubated without added T3 but did not increase [3H]uridine uptake in cells incubated with thyroid hormone. The PRL response to TRH was substantially inhibited 12 h after the addition of T3, and the uridine uptake response was completely blocked in 8 h. Two other stimuli of PRL secretion, sodium butyrate and isobutylmethylxanthine, were effective in the presence or absence of T3. Thyroid hormone did not reduce the specific binding of either [125I-Tyr1]somatostatin or [125I]iodoepidermal growth factor. Somatostatin decreased the secretion of GH and PRL by pituitary tumor cells grown with or without T3. The data show that the effects of thyroid hormones on TRH receptors are specific and suggest that regulation of receptor concentrations may be the direct cause of thyroid hormone regulation of pituitary responsiveness to TRH.