We investigated whether the balance between endothelium-derived relaxing factors (EDRFs) and endothelium-derived contracting factors (EDCFs) might be altered in mesenteric arteries from aged Otsuka Long-Evans Tokushima Fatty rats (OLETF) (a type 2 diabetic model) [vs. age-matched control Long-Evans Tokushima Otsuka rats (LETO)]. ACh-induced relaxation was impaired in the OLETF group and a tendency for the relaxation to reverse at high ACh concentrations was observed in both groups. This tendency was abolished by indomethacin. The NO- and/or EDHF-mediated relaxation, and the protein expressions of phospho-eNOS (Ser1177) and extracellular superoxide dismutase were also reduced in OLETF. An ACh-induced contraction was observed at higher ACh concentrations in the presence of N^G-nitro-L-arginine (L-NNA), but was greater in OLETF. This contraction in OLETF was reduced by cyclooxygenase (COX) inhibitors and by prostanoid-receptor antagonists. The ACh-induced productions of TXA₂ and PGE₂ were greater in OLETF than in LETO, as were the mesenteric artery COX-1 and COX-2 protein expressions. Moreover, tert-butyl hydroperoxide (t-BOOH) (membrane-permeant oxidant) induced a concentration-dependent contraction that was greater in OLETF. The t-BOOH-mediated contraction was increased both by L-NNA and by endothelium removal in LETO, but not in OLETF, suggesting that a negative modulatory role of the endothelium was lost in OLETF. These results suggest that an imbalance between EDRFs and EDCFs may be implicated in the endothelial dysfunction seen in aged OLETF mesenteric arteries, and may be attributable to increased oxidative stress.