Isolated perfused lung systems are commonly used to assess lung function in experimental studies. Assessment of hemodynamics and gas-exchange function in these systems is limited by the availability of venous blood. This study describes and validates a rat lung perfusion circuit in which a double-lung block ventilated with a hypoxic gas mixture [inspired O2 fraction (FIO2) 0.04; inspired CO2 fraction 0.08; deoxygenator (Deoxy) block] is used to provide blood with blood gases that are similar to mixed venous values to perfuse a study lung (FIO2 0.21; left lung only). This allows extended assessment of hemodynamics and gas exchange. Fifty adult male Wistar rats (300-400 g) were used as double-lung donors. Twenty-five perfusions (of both Deoxy and study lungs) were performed in four protocols (groups 1-5; n = 5). In protocol 1 (group 1), we tested whether exposure to room air affects the gas composition of the blood in the system. We found that the gas composition of the venous reservoir blood was identical to that of the blood entering the study block. In protocol 2, the effect of perfusion time and perfusion flow rate on the stability of the system was assessed. Lungs were perfused at 4 and 12 ml/min (groups 2 and 3, respectively), and the procedure was discontinued if edema or a marked decline in hemodynamics or gas-exchange function was observed. Pulmonary function was excellent and remained stable for 3 (at 12 ml/min) and 5 h (at 4 ml/min). In protocol 3, we examined whether hypoxic ventilation in the Deoxy lungs affects the stability of the system. Despite the low FIO2 used in the Deoxy lungs, the mean pulmonary arterial pressure-to-blood flow relationships in the study and Deoxy lungs were similar. Finally, in protocol 4, perfusion of a damaged study lung did not impair the function of the system. We conclude that this model permits reliable assessment of pulmonary function in rats under controlled ventilation and perfusion conditions. The use of a Deoxy double-lung block simplifies the perfusion apparatus and eliminates the main cause of instability of other systems that use an anesthetized host animal to provide venous blood.