Glucose metabolism was stimulated in isolated perfused rat lungs by perfusion with 2,4-dinitrophenol (DNP), an uncoupler of oxidative phosphorylation or phenazine methosulfate (PMS), an artificial hydrogen acceptor. Lungs were ventilated with 95% O2:5% CO2 and perfused with Krebs-Ringer bicarbonate buffer pH 7.4 containing 5.5 mM [U-14C, 5-3H]glucose for 100 min. In control lungs, 45% of total glucose 14C was recovered as perfusate lactate plus pyruvate (L+P) and 24% as 14CO2. Perfuson with 0.8 mM DNP resulted in a 102% increase in 14CO2 production and a 98% increase in L+P, associated with a fall in tissue ATP/ADP ratio and decreased incorporation of glucose carbons into lipids. Perfusion with 8 muM PMS resulted in a 116% increase in 14CO2 production but no change in L+P; the lactate-to-pyruvate ratio in the perfusate was reduced to 4 from a control value of 10, and tissue adenine nucleotide levels were unchanged. In all experiments, 3H was mainly recovered as 3H2O in the perfusate. These data demonstrate the pattern of response of the isolated lung preparation to uncoupling of oxidative phosphorylation and to alterations in cytoplasmic redox state. The results suggest that the isolated lung preparation is metabolically intact and suitable for study of metabolic control processes.