Previous work has demonstrated that 10 wk of exercise training enhances the responsiveness of rat abdominal aortas to acetylcholine (ACh), an endothelium-dependent vasodilator. The purpose of this study was to determine the time course for this training-induced adaptation of vascular endothelium. Additionally, the contribution of the cyclooxygenase and nitric oxide synthase mechanisms to the enhanced endothelium-mediated relaxation were examined. Male rats were divided into sedentary (SED) and exercise groups. Exercised animals were further subdivided into postexercise (POST-EX), 1 DAY, 1 WK, 2 WK, 4 WK and 10 WK groups. Exercise consisted of treadmill running at 30 m·min-1 (15° incline) for 1 h·d-1 (5 d·wk-1 for the 1 WK, 2 WK, 4 WK, and 10 WK groups). Maximal vasodilator responses induced by 10-4 M ACh (10-7 M norepinephrine preconstriction) were determined on abdominal aortic ringsin vitro immediately after a single exercise bout in POST-EX rats and 24 h after a single bout of exercise in 1 DAY animals. Maximal 10-4 M ACh-induced dilation of aortas from 1 WK, 2 WK, 4 WK, and 10 WK animals was determined 24 h after the last exercise bout. Soleus muscle citrate synthase activity was greater in 2 WK (31 ± 1μmol·min-1·g wet wt-1), 4 WK (34 ± 2), and 10 WK (36 ± 1 μmol·min-1·g wet wt-1) rats than in SED (27 ± 1 μmol·min-1·g wet wt.-1) animals. Maximal ACh-induced relaxation was greater in aorta from 4 WK (72 ± 2%) and 10 WK (79 ± 1%) rats than SED (61± 2%) rats. ACh-mediated dilatory responses remained enhanced in the presence of the cyclooxygenase blocker indomethacin (10-5 M), but were abolished by the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (300 μM). In addition, the expression of endothelial nitric oxide synthase (ecNOS) protein in aortas from 4 WK (P = 0.057) and 10 WK (P < 0.05) rats was greater than in aortas from SED animals. These data indicate that the enhanced endothelium-dependent, ACh-mediated dilation of the rat aorta is present by 4 wk of endurance exercise training. This adaptation appears to be mediated primarily through the nitric oxide synthase pathway and is associated with an increased expression of ecNOS.