N-Acetyltransferase (NAT) activity and melatonin content were determined in pineal glands obtained during the proestrous stage of the rat estrous cycle. In control animals, both pineal NAT activity and melatonin levels were reduced during the first hours of proestrous night, as compared with day-time proestrous levels. The administration of antiprogestagen RU486, in a dose which blocked the preovulatory rise of serum luteinizing hormone and follicle-stimulating hormone levels, failed to prevent the reduction of pineal melatonin synthesis associated with the ovulatory events at proestrous night. These results suggest that estradiol, but not progesterone, might be the ovarian hormone responsible for the inhibition of pineal melatonin synthesis observed in the normal cycling female rat during proestrous night.