In an attempt to delineate the immunological alterations that may occur following treatment with estrogen, groups of C57BL/6 mice were treated with 75 mg/kg body weight of β-estradiol-17-valerate (E2) or the vehicle. The thymus from these mice were harvested on days 1, 4 and 7 following treatment. The thymocytes from E2-treated mice when cultured in vitro for 24 h, showed increased levels of apoptosis when compared to controls. The apoptosis was demonstrable by both TUNEL assay and Annexin V/propidium iodide (PI) staining. Also, thymic atrophy and increased apoptosis of thymocytes when cultured in vitro were seen when lower doses of E2 (5 mg/kg) were administered. The thymus from E2-treated mice on days 4 and 7 also showed a decrease in the percentage of CD4+CD8+ (DP) T cells and an increase in the percentage of CD4-CD8- (DN), CD4+ and CD8+ T cells. However, the total cellularity of all T cell subsets in the thymus was decreased following E2 treatment. Earlier studies from our laboratory and elsewhere have demonstrated that in thymocytes undergoing apoptosis, there is increased expression of surface markers including CD3, αβTCR and CD44 with a simultaneous decrease in the expression of J11d. Similar changes were observed in thymocytes from mice on days 4 and 7 following E2 treatment. These data therefore confirmed that the thymocytes were indeed undergoing apoptosis following E2 treatment. Together, our studies suggest for the first time that estrogen may induce thymic atrophy by triggering apoptosis.