Pentose cycle activity was estimated in isolated rat lungs under varying conditions of glucose oxidation. Lungs were perfused for 100-120 min with a Krebs-Ringer-bicarbonate buffer, pH 7.4, containing 1-14C- or 6-14C-labeled glucose and ventilated with 95% O2:5% CO2. Based on 14C specific yields in either 14CO2 or perfusate lactate plus pyruvate, pentose cycle flux in control lungs was 5.3 mumol of glucose per hour per gram dry weight (11-12% of glucose utilization). Pentose cycle activity was unaltered by perfusion with 0.8 mM 2,4-dinitrophenol. Perfusion with phenazine methosulfate, an artificial hydrogen acceptor, resulations of the pentose cycle based on 14C yields in tissue lipids (both the fatty acid and deacylated fractions) gave values 2-3 times higher than measurements based on 14CO2. This study indicates that pentose cycle activity in the lung accounts for a significant fraction of glucose utilization and this pathway readily responds to metabolic perturbation.