T o determine whether the adventitia that surrounds pulmonary vessels acts as a barrier specific to nitric oxide, special lucite chambers were constructed to measure the force of contraction of rabbit pulmonary artery rings in which the endothelial or adventitial surfaces could be preferentially exposed to nitric oxide (NO), carbon monoxide (CO), or sodium nitroprusside (SNP). Delivery of NO to the endothelial and adventitial surfaces of preconstricted vessels produced markedly different concentration-response curves with maximal relaxations of 89 +/- 3 and 11 +/- 9%, respectively. In contrast, relaxations induced by both CO and SNP did not differ significantly between endothelial and adventitial exposure to these agents. Placement of a layer of pericardium onto the endothelial surface eliminated relaxation to the endothelial delivery of NO but not to CO. We conclude that the pulmonary vascular response to NO displays a striking sidedness which is not observed either with CO, another gas of similar molecular weight, or with SNP, both of which cause relaxation by stimulating guanylate cyclase. The elimination of NO but not CO relaxations with a layer of pericardium may indicate that the adventitia acts as a barrier specific to NO. This directionality of effect provides evidence for a highly localized regulation of pulmonary vascular tone by endothelial cell NO and also indicates that extravascular NO may have limited access to pulmonary vascular smooth muscle.