The circadian rhythm of prolactin secretion was assessed in postmenopausal women before and after daily administration of ethinyl estradiol for 4 weeks given in the morning (AM) or evening (PM) or in combination with a synthetic progestin, 17-medroxyprogesterone acetate. In response to AM ethinyl estradiol, mean circulating levels of serum prolactin during sleep and awake phases of the day rose 2.5-fold compared to untreated control values. The sleep/wake ratios for serum prolactin were comparable prior to and after AM administration of ethinyl estradiol. Altered time of administration, PM ethinyl estradiol or concomitant ethinyl estradiol and progestin treatment produced similar increases in mean prolactin levels, which were not different from the level observed during AM administration of ethinyl estradiol. These findings indicate that estrogen replacement therapy, in a dosage recommended for treatment of the menopause, stimulated a sustained rise in serum prolactin over 24 hours. This enhanced release of prolactin was not influenced by the time of administration of estrogen or concomitant administration of 17-medroxyprogesterone acetate.