The gender difference in cardiovascular disease has been partly attributed to higher androgenic hormone levels. Although testosterone in women may not affect lipids, it remains unknown whether it negates favorable estrogenic effects on endothelial function. We have investigated the effects of testosterone implant therapy on arterial reactivity encompassing endothelial-dependent and -independent vasodilation in women using hormone replacement therapy (HRT). B-mode ultrasound measurements of resting brachial artery diameter, following reactive hyperemia [endothelium-dependent flow-mediated dilation (FMD)] and following glyceryl trinitrate (GTN) (endothelium-independent dilation), were recorded in 33 post-menopausal women stabilized on HRT (>6 months), at baseline, and 6 weeks after a testosterone implant (50 mg), with 15 postmenopausal nonusers of HRT serving as controls. In the brachial artery, baseline resting diameter was similar (0.40 ± 0.01 vs. 0.41 ± 0.01 cm, P = 0.5). In the treated group, testosterone levels increased (0.99 ± 0.08 to 4.99 ± 0.3 nmol/L, P <0.001), associated with a mean 42% increase in FMD (6.4% ± 0.7 to 9.1% ± 1.1, P = 0.03). The control group did not change (8.1% ± 1.4 to 5.6% ± 1.0, P = 0.4). ANOVA of repeated measures (P = 0.04) and mean change (P = 0.02) in FMD both demonstrated significantly greater improvement with testosterone compared with controls. GTN induced vasodilation increased with testosterone treatment (14.9% ± 0.9 to 17.8% ± 1.2, P = 0.03). Our preliminary data indicate that parenteral testosterone therapy improves both endothelial-dependent (flow-mediated) and endothelium-independent (GTN-mediated) brachial artery vasodilation in postmenopausal women using long-term estrogen therapy. The mechanisms underlying these potentially beneficial cardiovascular effects require further investigation.