Acute anoxia or severe hypoxia causes an initial transient contraction followed by marked relaxation of vascular tissues. We observed a spontaneous gradual sustained contraction of rat aortic rings following relaxation when hypoxia was prolonged. Deendothelialization as well as treatment of the endothelium-intact rings with nitric oxide synthase inhibitors or oxyhemoglobin abolished the late hypoxic contraction despite prolonged hypoxia. The prolonged hypoxia-induced sustained contraction was not affected by adenosine receptor blockade, cyclooxygenase inhibition, free radical scavengers, or the endothelin receptor antagonists. The ATP-sensitive K+ channel blocker glibenclamide abbreviated the duration of hypoxic relaxation and potentiated the magnitude of late hypoxic contraction. These data suggest that the late-sustained hypoxic contraction of arterial tissues is dependent on the presence of intact functional endothelium. Activation of ATP-sensitive K+ channels may participate in the genesis of hypoxic relaxation. However, cyclooxygenase products, free oxygen radicals, adenosine, and endothelin are not involved in the regulation of hypoxia-mediated events in rat aortic rings.