Using borate buffer a substance that suppresses the ovulation induced with PMS and HCG in immature mice was obtained from acetone-defatted bovine pineal powder by an extraction method similar to that used for an extraction of a gonadotropin-inhibiting substance in urine. This gonadotropin inhibitor in the pineal powder differs from melatonin or arginine vasotocin and seems to be different from the water soluble antigonadotropic substance which has been isolated from the bovine and ovine pineal. Partial purification of the gonadotropin inhibitor was accomplished by a Sephadex G-100 column.