Melatonin administration has been shown to delay sexual maturation in male rats, through an action which involves decreased binding of gonadotropin-releasing hormone (GnRH) in the pituitary and lower pituitary gonadotropin contents. It has been suggested that melatonin must act at a level higher than the pituitary to provoke these effects, but a direct action of melatonin on the pituitary has not been excluded. Using a cell culture system, the gonadotroph responsiveness to GnRH was studied. Pituitaries were obtained either from rats chronically treated with melatonin and showing delayed sexual maturation, or from control rats. In vitro luteinizing hormone and follicle-stimulating hormone response to GnRH was significantly lower when pituitaries were obtained from melatonin-treated rats. However, this diminished response was directly proportional to the amount of gonadotropin contents in cells, so that relative responsiveness, calculated as the amount of gonadotropins released in relation to the gonadotropin content was similar in cells from control and melatonin-treated rats. It is concluded that the effect of melatonin on the pituitary of male rats results from a decrease of gonadotroph growth or gonadotropin synthesis, as a consequence of a change located at the hypothalamic rather than at the pituitary level. This conclusion is further supported by results showing that melatonin added directly in culture medium prior to GnRH does not modify the pituitary responsiveness to GnRH.