Immature female rats were induced to ovulate 72 h after an injection of pregnant mare serum (PMS). Melatonin, a compound synthesized in the pineal gland, was injected by three different routes: subcutaneously, intravenously, and intraocularly. In experiment 1, 10 µg of melatonin, injected at 12, 2, and 4 p.m. on day 2 after PMS, was ineffective in preventing ovulation, whether injected subcutaneously or into the eyeball. However, 100 µg of melatonin placed into the eyeball reduced the number of rats ovulating, as well as ovarian weights, relative to controls. Uterine weights of these rats were increased compared to controls. In experiment 2, the intraocular injection of 100 µg of melatonin inhibited ovulation in the greatest number of rats, compared to the intravenous or subcutaneous routes. Experiment 3 was a repeat of experiment 2, with comparable results. In experiment 4, with a dosage of 75 µg of melatonin, the intraocular administration appeared to be the most effective route for inhibiting ovulation. In experiment 5, subcutaneous administration of 50 µg of melatonin was completely ineffective in blocking ovulation or altering ovarian weights. The intraocular injection, however, significantly reduced ovarian weights.