We tested the hypothesis that exercise (Ex) training attenuates hypercholesterolemiainduced impairment of endothelium-dependent relaxation (EDR) in brachial (Br) arteries of adult male pigs by enhancing nitric oxide (NO)-mediated EDR. Adult male pigs were fed a normal fat (NF) or high fat/cholesterol (HF) diet for 20 weeks. Four weeks after the diet was initiated, pigs were trained or remained sedentary (Sed) for 16 weeks yielding four groups: 1) NF-Sed, 2) NF-Ex, 3) HF-Sed, and 4) HF-Ex. EDR of Br artery rings was assessed in vitro with acetycholine (ACh) and bradykinin (BK). ACh- and BKinduced relaxation was not impaired by HF; however, relaxation responses were enhanced by Ex in NF and HF arteries. To determine the mechanism(s) by which Ex improved EDR, ACh- and BK-induced relaxation was assessed in the presence of LNAME (to inhibit nitric oxide synthase), indomethacin (Indo; to inhibit cyclooxygenase), or L-NAME + Indo. ACh- and BK-induced relaxation was inhibited by L-NAME, and L-NAME + Indo, in all groups of arteries. Indo did not inhibit ACh-induced relaxation in any group but did inhibit BK-induced relaxation in HF-Ex arteries. In the presence of LNAME or L-NAME + Indo, ACh- and BK-induced relaxation in HF-Ex arteries remained greater than HF-Sed arteries. However, in the presence of Indo ACh-induced relaxation in HF-Ex arteries was no longer greater than in HF-Sed arteries. These results indicate that EDR is not impaired by hypercholesterolemia in Br arteries from adult male pigs; however, Ex improves EDR in HF Br arteries by enhancing production of endothelium-derived hyperpolarizing factor (EDHF) and/or prostacyclin (PGI₂).