Hypoventilation, associated with hypercapnic acidosis (HCA), may improve outcome in acute lung injury (ALI). We have recently reported that HCA per se protects against ALI. The current study explored whether the mechanisms of protection with HCA were related to acidosis versus hypercapnia. Because CO(2) equilibrates rapidly across cell membranes, we hypothesized that (1) HCA would afford greater protection than metabolic acidosis. We further hypothesized that (2) buffering HCA would attenuate its protection. Forty isolated perfused rabbit lung preparations were randomized to: control (normal pH, PCO(2)); HCA; metabolic acidosis; or buffered hypercapnia. After ischemia-reperfusion (IR) injury wet:dry ratio was greatest with control and buffered hypercapnia, and rank order of capillary filtration coefficient was: control approximately buffered hypercapnia > metabolic acidosis > HCA. Isogravimetric pressure reduction was greatest with buffered hypercapnia. Despite comparable injury, pulmonary artery pressure elevation was less with buffered hypercapnia versus control. In vitro xanthine oxidase (XO) activity depended on pH, not PCO(2). We conclude that: (1) HCA and metabolic acidosis are protective, but HCA is the most protective; (2) buffering HCA attenuates its protection; (3) buffering HCA causes pulmonary vasodilation; (4) because metabolic acidosis and HCA similarly inhibit in vitro XO activity, the differential effects cannot be explained solely on the basis of extracellular XO activity.