We quantitated the ability of intratracheally administered liposome- encapsulated antioxidant enzymes to reduce reactive oxygen species injury to the pulmonary microvasculature. Cationic liposomes containing 3,500 U of Cu,Zn superoxide dismutase (Cu,Zn SOD) and 3,124 U of catalase were instilled into rabbits. The animals were killed 2-72 h later and their lungs were removed and perfused with Krebs Ringer with 5% wt/vol of fat-free bovine serum albumin. The pulmonary filtration coefficient (K(f,c)) was measured before and after adding 500 μM xanthine and 5 mU/ml xanthine oxidase (XO) into the lung perfusate. Two hours after a single intratracheal instillation of liposome-entrapped Cu,Zn SOD and catalase, lung antioxidant enzyme activities were 34 and 125% higher than the corresponding control values, remained virtually unchanged for up to 8 h postinstillation, and then decreased, reaching baseline values between 24 and 72 h. Addition of xanthine and XO into the lung perfusate of uninstilled rabbits, or rabbits that received liposomes with inactivated enzymes, caused a 100% increase in K(f,c) (control value: 2 ± 0.12 ml · min-1 · cmH2O-1 per 100 g dry lung weight). On the other hand, K(f,c) values of rabbits lungs instilled with liposome-encapsulated active Cu,Zn SOD and catalase and challenged with xanthine and XO 8-24 h later remained at baseline levels. Instillation of liposomes containing either enzyme was equally effective in preventing the increase in K(f,c), indicating that both superoxide anions and hydrogen peroxide were necessary for the initiation of injury. We concluded that intratracheal instillation of liposome-encapsulated antioxidant enzymes causes a transient increase of lung antioxidant enzyme levels which protects the pulmonary microvasculature from free radical-initiated injury.