ABSTRACT This study investigated the effect of oestrogen exposure and exercise on caspase-3 activity, a measure of apoptosis, in lymphocytes from the thymus, spleen, and lymph nodes in ovariectomized mice. Fifty-nine female B6D2F1 mice were randomized to hormone and exercise conditions. Hormone treatment consisted of implantation with oestradiol pellets (0.72 mg oestradiol) or placebo pellets (0 mg) for 21 days following bilateral ovariectomy (OVX). Exercise consisted of a single treadmill exercise bout (26 m min−1, 6° slope, 90-min) or sedentary condition. Mice were killed and the thymus, spleen and lymph nodes were removed for the determination of caspase-3 expression by enzyme-linked immunosorbent assay (ELISA), serum oestrogen levels by RIA, and tissue weights. Body weights were monitored throughout the study. In the thymus, oestrogen exposure, exercise and both treatments together were associated with higher caspase-3 activity (P < 0.05) and lower thymus weights (P < 0.05). In contrast, oestrogen exposure and exercise treatment were not associated with greater caspase-3 activity or change in tissue weight in secondary lymphoid tissues (spleen, lymph nodes). Oestrogen-replaced OVX mice had a higher concentration of plasma oestradiol than placebo OVX mice (P < 0.05). Conclusion: The results suggest that oestrogen and treadmill exercise are associated with greater apoptosis, as measured by caspase-3 activity, in the thymus but not in the spleen or lymph nodes. Clinical studies will be necessary to determine if women who take oestrogen have higher rates of apoptosis in primary lymphoid tissues and the significance of thymocyte apoptosis for maintenance of cellular immune function during the post-menopausal years.