Administration of 1.25–5 mg of melatonin dissolved in 10% methanol in saline by intracardiac route during the critical period of proestrus (2–4 PM) resulted in complete inhibition of ovulation during presumptive estrus. All animals receiving the vehicle shed an average of ten ova. The inhibition can be overcome by iv injection of 10 ¼g luteinizing hormone (LH) thereby suggesting that melatonin is not acting at the ovarian level. Results of radioimmunoassay of serum LH levels in melatonin and vehicle—treated animals indicate that multiple injections of melatonin prevented the release of LH and inhibited ovulation. However, the same doses given before or after the critical period on the day of proestrus did not affect the incidence of ovulation. It is concluded that melatonin, at the dose levels used affects directly the ovulatory surge of gonadotropin and consequently inhibits ovulation. These results further support the concept that the pineal gland exerts control over reproductive functions via an inhibiting action of melatonin on the release of hypophyseal gonadotropins. (Endocrinology92: 333, 1973)