The aim of this study was to investigate the effects of lipid and organic hydroperoxides on vasomotor activity of isolated rat superior mesenteric arteries. Hydroperoxides did not elicit measurable responses in unstimulated (quiescent) mesenteric arteries. Contractile responses to potassium, however, were significantly potentiated by 13-(s)-hydroperoxylinoleic acid (range 3–54 μM). Potentiation of potassium responses by linoleic acid (18:2) and linolenic acid (18:3) was increased by pretreatment of the fatty acids with lipoxygenase (p < .01). Lipoxygenase alone had no contractile effects. Lipoxygenase-treated 18:2, tert-butyl hydroperoxide, and hydrogen peroxide augmented contractile responses to phenylephrine, but to a lesser degree than corresponding augmentation of potassium responses. Contractile responses to lipoxygenase-treated 18:3 were blunted by vitamin E (p < .02) and by nitroblue tetrazolium (p < .02), whereas catalase and mannitol had no effects, implicating lipid free radicals in the contractile response. Responses to lipid hydroperoxides were not significantly altered by prostaglandin inhibitors. Endothelial cell denudation significantly enhanced the contractile responses elicited by 13-(s)-hydroperoxylinoleic acid (p < .05), indicating that lipid hydroperoxides enhance agonist-induced contractions by a direct effect on the smooth muscle. These results support a hypothesized link between lipid peroxidation and development of altered vascular function. They further suggest that the vascular endothelium may play an important role in regulation of vasomotor responses to lipid hydroperoxides.