The relations between the circadian rhythms of melatonin and prolactin, and the effect of melatonin infused into the third ventricle or the mediobasal hypo-thalamus (MBH) on prolactin secretion and dopamine (DA) release were studied in anestrous ewes under increasing daylength conditions. The decreased amplitude and duration of nocturnal melatonin secretion were accompanied with changes in the daily pattern of prolactin secretion. Marked peaks of prolactin occurred after sunrise and melatonin decreases in February, as well as significant evening or nocturnal peaks under long day conditions between April and July. Melatonin infused into the third ventricle evoked an abrupt increase in the concentration of prolactin after 30 min, and the enhanced prolactin level was significantly higher than during the control infusion (range from 204 ± 75 to 248 ± 48 ng/ml vs. 128 ± 68 to 149 ± 93 ng/ml, mean ± SD). The concentrations of DA in MBH perfusates decreased during perfusion of melatonin but to a degree similar to that noted in untreated ewes. These data suggest that short-term infusions of melatonin stimulate the secretion of prolactin in the ewe under increasing daylength conditions, and that this effect is not mediated by changes in DA release.