A new method for perfusion of rat lungs in situ was developed for metabolic studies. The pulmonary circulation was cannulated without contacting the lungs, which remained in the thoracic cage. Perfusion was continued for up to 4 h with Krebs-Henseleit bicarbonate buffer, equilibrated with 95% O2- 5% CO2 and containing 4.5% bovine serum albumin, 5.6 mM glucose, and levels of amino acids normally found in rat plasma. At an arterial pressure of 20 cmH2O flow remained constant (10.9 ml/min.100 g body wt) and appeared evenly distributed among the lobes. Tidal volume was 1 ml/100 g body wt (72/min); positive end-expiratory pressure was 2 cmH2O. The preparation remained stable and metabolically active for 4 h, as evidenced by a minimal decline in dry-to-wet weight ratio, constant levels of ATP and glycogen, a high ratio of glucose uptake to lactate production, and a linear rate of incorporation of [14C]phenylalanine into protein. The lungs were unaffected when perfusate oxygen was reduced to a more physiological level (20% O2-75% N2-5% CO2). In the presence of 95% N2-5% CO2 dry-to-wet weight ratio, ATP, glycogen, and amino acid incorporation decreased, while lactate production doubled.