The purpose of these studies was to determine whether diurnal rhythms in serotonin (5HT) activity are detectable in individual hypothalamic nuclei of ovariectomized rats and whether estradiol induces specific rhythms of 5HT which may be necessary to cyclic release of LH and/or PRL. Young (3- to 4-month old) rats were bilaterally ovariectomized and 7 days later half the animals received Silastic estradiol capsules. Two days later groups were again divided: half the animals in each group were killed at 0800, 1200, 1800, and 2400 h. The remaining animals received pargyline (75 mg/kg body weight, ip) at these times and were killed 10 min later. The median eminence (ME), suprachiasmatic nucleus (SCN), medial preoptic area (MPN), arcuate nucleus (AN), and globus pallidus (GP) were microdissected and assayed for 5HT by HPLC using electrochemical detection. A diurnal rhythm in 5HT turnover was found in the SCN, MPN, and AN of ovariectomized rats. 5HT turnover in these areas was significantly higher during the light hours (0800, 1200, and 1800 h) compared to the dark phase (2400 h). The ME and GP of ovariectomized rats did not exhibit a diurnal rhythm in 5HT activity. Exposure to estrogen altered the pattern of 5HT activity in all hypothalamic areas examined. In the ME, treatment with estradiol increased 5HT turnover at 1200 h, just before the predicted LH and PRL surge, and suppressed activity at all other times. In the SCN, estradiol reversed the 5HT rhythm: turnover was low during the light hours and high during the dark. In the AN and MPN, estradiol treatment increased 5HT activity and abolished the diurnal rhythm. 5HT activity in the GP was not altered by exposure to estrogen. We conclude from these data that specific brain nuclei exhibit diurnal rhythms in 5HT turnover and that the patterns of 5HT activity in specific hypothalamic nuclei exhibit individual and unique responses to the presence of estrogen. These data suggest that the estradiol-induced diurnal pattern of 5HT activity may be necessary for the induction of cyclic release of LH and/or PRL.