An experiment was performed on 21-day-old male rats to determine the combined effects of pinealectomy, constant light and darkness, and intraperitoneal (i.p.) thyrotropin-releasing hormone (TRH) on pituitary and plasma radioimmunoassayable thyrotropin (TSH), serum thyroxine (T<sub>4</sub>), and pituitary, thyroid and body weights at the age of 25 days. In saline-treated rats, pinealectomy or constant illumination decreased pituitary TSH and increased plasma TSH and serum T<sub>4</sub>. Constant darkness with an intact pineal, however, decreased all 3 of these parameters. When i.p. TRH was injected all rats showed an increase in plasma TSH as compared to saline-treated controls. Another study was performed on 25-day-old male rats to determine the effects of intraventricular administration of melatonin (MEL) alone, and intraventricular MEL plus i.p. TRH on pituitary and plasma TSH. MEL decreased plasma TSH levels as compared to non-treated and saline-treated controls, whereas the concurrent administration of TRH obviated the effect of MEL and increased plasma TSH levels above those of the control animals. The results are interpreted as indicating that the inhibitory effect of the pineal in dark-reared, sham-operated prepuberal male rats is exerted at the level of hypothalamic secretion of TRH.