Orchidectomy has been reported to decrease concentrations of thyrotrophin (TSH) in the circulation of male rats without affecting serum levels of thyroid hormones. To understand the mechanism underlying this observation, we have measured the effect of gonadal status on the in-vitro release of TSH-releasing hormone (TRH) by male rat hypothalamic fragments. Because hormone release rates can be affected by changes in the post-translational processing of the hormonal precursors, we have also studied the corresponding changes in the concentrations of TRH and TRH-Gly, a TRH precursor peptide in hypothalamus and pituitary, by radioimmunoassay. We observed a significant decline in the in-vitro release of TRH from incubated hypothalami 1 week after castration, which was quantitatively reversed by testosterone replacement. Concentrations of TRH and TRH-Gly in the posterior pituitary, on the other hand, which derive from neurones of hypothalamic origin, increased significantly with castration and were returned to the normal range by testosterone replacement. We conclude that the primary effect of testosterone is the stimulation of hypothalamic TRH release, resulting in the depletion of TRH and TRH precursors from TRH-containing neurones which project into the median eminence and posterior pituitary.