A melatonin-induced supersensitivity of the gonadotropin-secretory system to the negative feedback action of sex steroids is thought to be important to the timing of seasonal reproduction. However, little is known concerning this action of melatonin. In the present study the antigonadal action of melatonin in the anterior hypothalamus (AH) of the white-footed mouse, Peromyscus leucopus, was used to examine the neuroendocrine mechanism whereby melatonin enhances the sensitivity to sex steroid negative feedback. Mice received a melatonin-containing pellet in the AH for 14 weeks, at which time they were castrated and treated sc with a Silastic testosterone (T) capsule for 3 weeks. At the time of castration, weight of the testes and the concentration of T in the blood of mice with a melatonin pellet were greatly reduced compared to mice with a blank (melatonin-free) implant in the AH (P less than 0.01). In mice treated with melatonin the physiological dose of T significantly reduced the concentrations of LH in blood and pituitary (P less than 0.05). This dose of T, however, had little effect on LH in mice with a blank pellet in the AH. Melatonin in the AH markedly increased the content of gonadotropin-releasing hormone (GnRH) in the mediobasal hypothalamus (P less than 0.05) in mice treated with T; however, there was little effect of melatonin and/or T in any other region examined. Melatonin and T had little effect on the contents of immunoreactive beta-endorphin (B-EP) in the hypothalamus, but T alone increased the content of B-EP in the preoptic area. These results are evidence that melatonin and T act in concert to induce the reproductively-quiescent state by suppressing secretion of GnRH from the hypothalamus.