Although estrogens have been shown to be vasoactive hormones, the vascular effects of testosterone are not well defined. Little is known about the in vitro effects of testosterone on isolated arteries. The purpose of this experimental prospective study was to assess the direct effect of testosterone on isolated sheep coronary artery in vitro. We evaluated whether the vascular effects of testosterone were altered in the presence of endothelium or it changed in accordance to gender or the concentration of testosterone. Coronary arterial rings were precontracted by KCl (30 mM). After plateau contraction levels were reached, testosterones (10(-7)-10(-4)M) were added in a cumulative manner. The effects of testosterone were also tested in the presence of NOS inhibitor Nω-nitro-L-arginine methyl ester (L-NAME, 10-4M), and L-arginine (10(-4)M). In statistical analysis, Student's t-test was used for comparison between two groups and one-way ANOVA test for comparison among multiple groups. Testosterone relaxed sheep coronary artery. Testosterone-induced relaxation was dependent of sex [male endothelium (ME(+)): 24.48±4.13; -29.36±6.41; female endothelium (FE(-)): -3.02±1.04: p<0.05); (ME(+): -24.61±4.14; -24.48±4.13; -29.36±6.41; FE(-): -3.64±0.67: p<0.01); (FE(-):-4.91±0.67: p<0.001)] and endothelium (ME(+): -9.23±2.4; FE(+): -6.33±1.5; -8.43±0.49; -8.83±0.9: p<0.05, p<0.01). L-NAME decreased relaxations in the male (ME(+): 0.77±0.72; 0.086±0.012; 0.0768±0.083; 0.51±0.44: p<0.01, p<0.001) and female (FE(+): 0.0318±0.052; 0.52±0.49; 0.029±70.05: p<0.05, p<0.001) with endothelium groups but only female without (0.01±0.011; -1.14±0.59; -1.23±1.21: p<0.01, p<0.001) endothelium group. L-arginine decreased relaxations especially in the male with endothelium (ME(+): -1.7±0.91; -3.02±1.42; -2.51±1.46; -6.68±2.15; p<0.01, p<0.001) group and in part in the female with (FE(+):-1.73±0.83; p<0.05) or without (FE(-): -1.14±0.59; p<0.01) endothelium groups. Testosterone induces endothelium and sex-dependent relaxation on sheep coronary artery in vitro. Acute testosterone-induced coronary vasodilatation is mediated in part via endothelium-derived NO. Non-genomic mechanisms may be considered to play a role in the vasodilatory effect of testosterone especially in the female group.