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 b 2 -adrenoceptor (b 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 mg/kg per day (OvxCE), Ovx rats with testosterone 150 mg/kg per day (OvxCT), and Ovx rats with E 2 40 mg/kg per dayC testosterone 150 mg/kg per day (OvxCE/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 b 2 -AR antagonist ICI 118 551. We also determined the expression of b 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 b 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 b 2 -AR. The study highlights the potential therapeutic application for CVD in postmenopausal women.