We studied the effects of surfactant supplementation on the progression of lung injury in rabbits exposed to 100% O2 for 64 h and returned to room air for 24 h. At this time, rabbits not treated with surfactant exhibit a severe lung injury with hypoxemia, increased alveolar permeability to solute, decreased total lung capacity (TLC) and lung edema. For surfactant treatment, 125 mg of calf lung surfactant extract (CLSE), suspended in 6-8 ml of normal saline, were instilled intratracheally at 0 and 12 h posthyperoxic exposure. At 24 h postexposure, these CLSE-treated rabbits compared with saline controls had significantly higher amounts of lung phospholipids (34 ± 4 vs. 4.5 ± 0.6 μmol/kg body wt) and increased TLC (42 ± 2 vs. 27 ± 1 ml/kg), with significantly lower amounts of alveolar protein (36 ± 3 vs. 56 ± 3 mg/kg) and decreased lung wet weight-to-dry weight ratios (5.6 ± 0.1 vs. 6.3 ± 0.3). Surfactant supplementation also decreased the degree of lung atelectasis as reflected by the increase in arterial O2 partial pressure (Pa(O2)) after breathing 100% O2 for 20 min (Pa(O2) = 460 ± 31 vs. 197 ± 52 Torr). These findings indicate that instillation of exogenous surfactant mitigates the progression of hyperoxic lung injury in rabbits.