There have been contradictory reports suggesting that CO(2) may constrict, dilate, or have no effect on pulmonary vessels. Permissive hypercapnia has become a widely adopted ventilatory technique used to avoid ventilator-induced lung injury, particularly in patients with acute respiratory distress syndrome (ARDS). On the other hand, respiratory alkalosis produced by mechanically induced hyperventilation is the mainstay of treatment for newborn infants with persistent pulmonary hypertension. It is important to clarify the vasomotor effect of CO(2) on pulmonary circulation in order to better evaluate the strategies of mechanical ventilation in intensive care. In the present study, pulmonary vascular responses to CO(2) were observed in isolated rat lungs (n = 32) under different levels of pulmonary arterial pressure (PAP) induced by various doses of endothelin-1 (ET-1). The purposes of this study were to investigate (1) the vasodilatory effect of 5% CO(2) in either N(2) (hypoxic-hypercapnia) or air (normoxic-hypercapnia) at different PAP levels induced by various doses of endothelin-1, and (2) the role of nitric oxide (NO) in mediating the pulmonary vascular response to hypercapnia, hypoxia, and ET-1. The results indicated that (1) CO(2) produces pulmonary vasodilatation at high PAP under ET-1 and hypoxic vasoconstriction; (2) the vasodilatory effect of CO(2) at different pressure levels varies in accordance with the levels of PAP, the dilatory effect tends to be more evident at higher PAP; and (3) endogenous NO attenuates ET-1 and hypoxic pulmonary vasoconstriction but does not augment the CO(2)-induced vasodilatation.