17 beta-Estradiol (E2) was dissolved in the drinking water of female C57BL/6J mice and presented ad libitum. The oral administration of E2 produced expected responses in E2-sensitive target tissues. Vaginal smear cytology changed from the thin leukocytic smears characteristic of ovariectomized controls to thicker smears containing only cornified epithelial cells. Uterine weight and the specific activities of uterine glucose-6-phosphate dehydrogenase and alkaline phosphatase were elevated, while the postovariectomy elevation in serum luteinizing hormone (LH) was suppressed. However, oral E2 did not influence the specific activity of uterine acid phosphatase. During oral administration of E2 through the drinking water, serum estrone and E2 were elevated during the night and returned to low baseline levels during the day, in parallel with the circadian patterns of drinking. Similar transient elevations of serum E2 levels were observed after subcutaneous injections of E2. The oral administration of E2 has advantages over the widely utilized parenteral routes of E2 administration (i.e., injection or surgical implantation of E2-containing capsules), particularly for long-term experiments, and may be more analogous to the usual oral route of estrogen administration in women as contraceptives or as postmenopausal estrogen-replacement therapy.