It is not known where melatonin acts to influence the neuroendocrine axis of seasonally breeding mammals. However, since the pars tuberalis (PT) contains the highest density of melatonin receptors, this adenohypophyseal subdivision is a potential target. Gonadotropes are the only immunocytochemically detectable adenohypophyseal cell type of abundance in the PT. This study investigated whether melatonin could modulate basal and/or gonadotropin-releasing hormone (GnRH)-stimulated luteinising hormone (LH) secretion from the ovine PT and pars distalis (PD) in vitro. Tissue fragments from both pituitary areas were placed in separate chambers in a constant-environment perifusion system (37°C; 100 µl/min) and 10-min effluent fractions were collected, frozen and later assayed for LH. Sixty minutes prior to a GnRH challenge (10 min; 10 nM), melatonin (1 µM or 100 nM) was added to the perifusate of half the tissue fragments. GnRH increased (p < 0.01) LH output from both pituitary subdivisions. Melatonin attenuated (p < 0.05) the GnRH-induced increase in LH output from the PT but not from the PD. The physiological importance of this melatonin-attenuated PT LH is unknown but it may play a role in modulating the neuroendocrine reproductive axis.