Experiments were designed to localize the central sites of action of melatonin involved in the control of seasonal reproduction. Ewes were exposed to long days and received microimplants of melatonin in the preoptic area (n = 9), anterior hypothalamus (n = 4), dorsolateral hypothalamus (n = 4), or mediobasal hypothalamus (n = 12). The effects of implants were determined by comparison with control ewes (untreated or sham-operated, n = 10) and with ewes treated with an s.c. implant of melatonin (n = 8) or ewes subjected to short days (n = 8). All ewes were ovariectomized and treated s.c. with a silastic capsule of estradiol. Melatonin released in the preoptic area as well as in the anterior and lateral hypothalamus did not cause any difference as compared with the controls (no stimulation of LH secretion and no inhibition of prolactin secretion). In contrast, melatonin implanted in the mediobasal hypothalamus caused an increase in LH secretion in 7 of the 12 ewes on Day 53.0 ± 4.2 after implantation (mean ± SEM). Their response was not different compared with that of ewes treated s.c. with melatonin or exposed to short days either in terms of timing (Day 56.3 ± 6.2 and 59.5 ± 3.1, respectively, for controls) or of amplitude of the LH response. Similarly, melatonin caused only a reduction of prolactin secretion in the mediobasal, s.c., and short-day groups. It is concluded that the mediobasal hypothalamus or the surrounding tissues could be the sites of action of melatonin involved in the control of seasonal reproduction.