After menopause, the development of cardiovascular disease (CVD) is due not only to estrogen decline but also to androgen decline. This study examined the effects of either estradiol (E(2)) or testosterone replacement alone or E(2)-testosterone combination on isolated myocytes in ovariectomized (Ovx) rats subjected to ischemia/reperfusion (I/R). Furthermore, we determined whether the effects are associated with β(2)-adrenoceptor (β(2)-AR). Five groups of adult female Sprague-Dawley rats were used: Sham operation (Sham) rats, bilateral Ovx rats, Ovx rats with E(2) 40 μg/kg per day (Ovx+E), Ovx rats with testosterone 150 μg/kg per day (Ovx+T), and Ovx rats with E(2) 40 μg/kg per day+testosterone 150 μg/kg per day (Ovx+E/T). We determined the lactate dehydrogenase (LDH) release, percentage of rod-shaped cells and apoptosis of ventricular myocytes from rats of all groups subjected to I/R. Then, we determined the above indices and contractile function with or without a selective β(2)-AR antagonist ICI 118 551. We also determined the expression of β(2)-AR. Our data show that either E(2) or testosterone replacement alone or E(2) and testosterone in combination decreased the LDH release, increased the percentage of rod-shaped cells, reduced apoptotic cells (%), and combination treatment appeared to be more effective than either E(2) or testosterone replacement alone. ICI 118 551 abolished the effects of the three. Combination supplementation also enhanced the expression of β(2)-AR. We concluded that in Ovx rats, testosterone enhances E(2)'s cardioprotection, while E(2) and testosterone in combination was more effective and the protective effects may be associated with β(2)-AR. The study highlights the potential therapeutic application for CVD in postmenopausal women.