Studies from our laboratory have previously demonstrated sensitive and specific autoregulatory control systems for thyrotropin (TSH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) in the rabbit. Because our studies of LH autoregulation showed the feedback regulation acted directly at a pituitary level, the current studies were designed to investigate whether the TSH control system also acted at the pituitary level. Two species-specific TSH assays were employed; a rabbit TSH radioimmunoassay which showed little or no reaction to human TSH, and a human TSH radioimmunoassay which showed little or no reaction to rabbit TSH. Both in vivo and in vitro studies were performed. TRH (thyrotropin-releasing hormone) in doses of 2, 10, and 50 micrograms was injected as an intravenous bolus into thyroidectomized hypothyroid rabbits during continuous perfusion with highly purified human TSH (hTSH) or with saline. In these in vivo studies, TRH-stimulated rabbit TSH (rTSH) secretion was suppressed by hTSH perfusion compared with control saline perfusion. The effect of hTSH was studied in vitro by employing short-term cultured rabbit pituitary cells. When hTSH was added to the incubation medium, TRH-stimulated rTSH secretion was inhibited. From these studies, we conclude that one site of the autoregulatory control for TSH in the rabbit is at the pituitary level. These studies do not exclude a possible additional short-loop feedback control at an hypothalamic level, but such a site of action is not required to explain the autoregulatory phenomenon.