We studied endothelial dysfunction of the rabbit pulmonary artery following in vivo ischemia and reperfusion of the lung, and also investigated the mechanisms of endothelium-dependent relaxation in these arteries. Intrapulmonary arteries were isolated from rabbits subjected to ischemia and reperfusion of one lung. Percent relaxation values of sham-operated (i.e. nonischemic) pulmonary arteries to endothelium-dependent vasodilators acetylcholine (ACh) and A23187 were 72 ± 4 and 65 ± 4%, respectively, while relaxation to the endothelium-independent dilator NaNO<sub>2</sub> was 97 ± 1 %. The relaxation of control artery rings to ACh and A23187 were significantly decreased to 2 ± 1 and 5 ± 4%, respectively, following addition of Nω-nitro- L-arginine methyl ester, while relaxation following treatment with indomethacin or glybenclamide remained normal. Relaxation to NaNO<sub>2</sub> was not altered by pretreatment with any of the above compounds. Thus, pulmonary artery relaxation to the endothelium-dependent dilators ACh and A23187 appears to be mediated by the release of EDRF. Endothelium-dependent relaxation of pulmonary arteries from lungs exposed to 90 min of ischemia and 30 min of reperfusion remained essentially normal, while 90 min of ischemia followed by 60 min of reperfusion resulted in a significant decrease in endothelium-dependent relaxation to A23187 to 37 ± 7% (p < 0.05), whereas the response to ACh was reduced only to 57 ± 3% (not significant). 90 min of ischemia followed by 90 min of reperfusion resulted in significant attenuation of endothelium-dependent relaxation to both ACh (36 ± 4%) and A23187 (33 ± 7%). Intrapulmonary artery rings from rabbits given superoxide dismutase starting 15 min before reperfusion showed an improved relaxation to ACh and A23187(55 ± 5 and 58 ± 11%, respectively, at 90 min of reperfusion). Thus, in vivo ischemia and reperfusion of the rabbit lung results in significant endothelial dysfunction which worsens with increasing reperfusion time. Some of the endothelial dysfunction appears to be related to superoxide radical formation.