The present study evaluated the possible role of estrogens in generating the circadian rhythm of medial basal hypothalamus content at the time of puberty in female rats. Accordingly, changes in medial basal hypothalamus beta-endorphin (beta-EP) content were investigated in female rats, before and at puberty. Groups of intact or ovariectomized rats were studied after estradiol-benzoate or placebo treatment. The results showed that circadian rhythm of beta-EP content of medial basal hypothalamus is absent in prepubertal rats, while it appears at puberty, associated to a significant increase of beta-EP concentration. The primary involvement of steroids in generating this circadian rhythm was supported by the finding that estradiol-benzoate treatment caused a precocious appearance of beta-EP hypothalamic diurnal changes in prepubertal rats. Moreover, estradiol-benzoate replacement restored the loss of beta-EP nocturnal increase induced by ovariectomy in pubertal animals. Therefore, these data support the significant role of estrogen in inducing the circadian rhythm of beta-EP content in medial basal hypothalamus at the time of puberty in female rats.