Continuing investigation of pineal gland function indicates that the anti-gonadotrophic activity of this organ cannot be attributed solely to the postulated hormone melatonin, the concentration of which is negligible in the pineal body compared to quantities required to produce unequivocal physiological effects. A non-melatonin antigonadotrophic substance recently isolated from bovine pineal glands was further purified by organic solvent extraction, ultrafiltration and gel filtration. Studies of partial blockage of compensatory ovarian hypertrophy in unilaterally ovariectomized Charles River CD-1 mice indicated that this substance is significantly more potent than melatonin in this test system.