Flow-mediated dilatation (%FMD), an index of nitric oxide (NO)-mediated vasodilator function, is regarded as a surrogate marker of cardiovascular disease. Aging is associated with endothelial dysfunction, but underlying sex-related differences may exist and the effects of fitness and exercise on endothelial dysfunction in men (M) and women (W) are poorly understood. We compared %FMD of the brachial artery in 18 young [Y, 26 +/- 1 yr; 9 M and 9 W], 12 older fit (OF, 57 +/- 2 yr; 6 M and 6 W), and 16 older sedentary (OS, 59 +/- 2 yr; 8 M and 8 W) subjects. Glyceryl trinitrate (GTN) administration was used to assess endothelium-independent vasodilatation, and the FMD-to-GTN ratio was calculated to characterize NO dilator function in the context of smooth muscle cell sensitivity. Brachial %FMD in Y (7.1 +/- 0.8%) was significantly higher compared with OS (4.8 +/- 0.7%, P < 0.05), but not OF (6.4 +/- 0.7%). Differences between Y and OS subjects were due primarily to lower FMD in the OS women (4.3 +/- 0.6%). OS women exhibited significantly lower FMD-to-GTN ratios compared with Y (P < 0.05) and OF women (P < 0.05), whereas these differences were not apparent in men. Exercise training improved brachial artery NO dilator function (FMD-to-GTN ratio) after 24 wk (P < 0.05) in OS women, but not men. These findings indicate that maintaining a high level of fitness, or undertaking exercise training, prevents the age-related decline in the brachial artery vasodilator function evident in women. In OS men, who had relatively preserved NO dilator function, no training adaptations were observed. This study has potential implications for the prevention of conduit artery endothelial dysfunction in men and women.