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 Zucker diabetic fatty (ZDF) rats, we examined the effects of AA in small mesenteric arteries preconstricted with endothelin. In ZDF rat mesenteric arteries, 1 microM AA produced only one-third the amount of dilation as in vessels from lean control animals. In lean control rats, the effect of AA was significantly and predominantly inhibited by the lipoxygenase inhibitors baicalein and cinnamyl-3,4-dihydroxy-cyanocinnamate (CDC). However, baicalein and CDC had no effect on AA-mediated dilation in ZDF rat mesenteric arteries. The major [3H]AA metabolite produced by isolated mesenteric arteries in both lean and ZDF rats was 12-hydroxyeicosatetraenoic acid (12-HETE), but the amount of [3H]12-HETE produced by ZDF rat vessels was only 36% of that of control vessels. 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 rat mesenteric arteries. Immunofluorescence labeling showed strong nitrotyrosine signals in ZDF rat mesenteric arteries that colocalized with 12-lipoxygenase in endothelium, and 12-lipoxygenase coprecipitation with anti-nitrotyrosine antibodies was enhanced in ZDF rat vessels. We conclude that AA-mediated relaxation in ZDF rat 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.