The effects of estradiol on pineal function haveinsofar been studied by administering supramaximal, non-physiological doses of the hormone. The present experiments were undertaken to assess estradiol-induced changes on two parameters of pineal function, i.e., hydroxyindole-O-methyl transferase (HIOMT) activity and 3H-leucine incorporation into pineal proteins. Estradiol benzoate (0.5–3.2 µg) increased, 24 h later, 3H-leucine incorporation to pineal proteins almost 2-fold without significant changes in the protein content of the gland. A similar stimulatory effect was observed on pineal HIOMT, i.e., estradiol benzoate (0.05–1 µg/day for 3 days) enhanced melatonin synthesis and reversed the inhibitory effects of ovariectomy on HIOMT activity. In intact rats, HIOMT activity reached its maximum at the day of estrus. Increases in plasma estradiol occurring in proestrus may thus induce increases in pineal HIOMT activity 24 h later.