Arachidonic acid (AA) is a precursor of important vasoactive metabolites but the role of AA-mediated vasodilation in type 2 diabetes is not known. Using the Zucker Diabetic Fatty (ZDF) rats, we examined the effect of AA in small mesenteric arteries preconstricted with endothelin. In ZDF mesenteric arteries, 1 µM AA produced only one-third the amount of dilation as in vessels from Lean controls. In Lean controls, the AA effect was significantly and predominantly inhibited by the lipoxygenase inhibitors baicalein and cinnamyl-3,4-dihydroxycyanocinnamate (CDC). However, baicalein and CDC had no effect on AA-mediated dilation in ZDF mesenteric arteries. 12-Hydroxyeicosatetraenoic acid (12-HETE) was the major [³H]AA metabolite produced by isolated mesenteric arteries in both Lean and ZDF rats, but the amount of [³H]12-HETE produced by ZDF vessels was only 36% of that in controls. In addition, 12-HETE produced similar amounts of dilation in Lean and ZDF rat mesenteric arteries. Immunoblot analysis showed an 81% reduction in 12-lipoxygenase protein in ZDF mesenteric arteries. Immuofluorescence labeling showed strong nitrotyrosine signals in ZDF mesenteric arteries that co-localized with 12-lipoxygenase in the endothelium, and 12-lipoxygenase co-precipitation with anti-nitrotyrosine antibodies was enhanced in ZDF vessels. We conclude that AA-mediated relaxation in ZDF small mesenteric arteries is impaired due to reduced 12-lipoxygenase protein and activity. Increased oxidative stress and nitration of 12-lipoxygenase may underlie the impairment of AA-mediated relaxation in small mesenteric arteries of diabetic rats.