Because multiple substrates have been shown to play a role in the metabolic homeostasis of different tissues, a series of studies were initiated to examine the role of alternate substrates in the lung. In these studies, we measured rates of oxidation of glutamine, glucose, lactate and 3-hydroxybutyrate in fibroblasts isolated from d 19 fetal rat lungs by measuring the production of 14CO2 from labeled substrates and compared them with earlier studies of isolated Type II cells. The rate of glutamine oxidation was 16.04 nmol 14CO2 x mg protein(-1) x hr(-1) in the fibroblasts compared with 24.36 in Type II cells. Three-hydroxybutyrate had a rate of 10.75 in the fibroblasts and 14.9 in the Type II cells. Lactate oxidation in fibroblasts was similar to that of glutamine, with a rate of 18.49; however, in Type II cells the rate of lactate oxidation was significantly higher at 40.29. Glucose was oxidized at a rate significantly lower than the other three substrates. In the fibroblasts, that rate was 1.22 and in Type II cells it was 2.13. To examine the interactions of substrates normally found in the intracellular milieu, we measured the effect of unlabeled substrates as competitors on labeled substrate in the fibroblasts, similar to our studies with Type II cells that identified multiple metabolic compartments of energy metabolism in these cell populations. Glucose, but not lactate, inhibited the oxidation of glutamine, suggesting a compartmentation of tricarboxylic acid cycle activity rather than simple dilution by glucose. Glucose and lactate had reciprocal inhibition in the Type II cells. Our data suggest at least two separate compartments in developing lung cells for substrate oxidation: one for glutamine metabolism and a second for glucose metabolism. In summary, we have documented that glutamine and other alternate substrates are oxidized preferentially over glucose for energy metabolism in the d 19 fetal rat lung.