Using a sensitive technique measuring14CO2production from radiolabeled malate, we examined malic enzyme activity in both adult and newborn rat lung tissue and in L2 cells, a cell culture line of type II pneumocytes. Malic enzyme was present in both cytosolic and mitochondrial fractions. Time course experiments demonstrated a linear rate after the initial 10 min, up to 30 min. The optimal pH in the cytosolic fraction was 8.0, whereas maximal mitochondrial malic enzyme activity occurred at pII 7.0. The mitochondrial fraction exhibited biphasic kinetics over the 200-fold range of concentrations examined. The high-affinity Km was 0.16 mmol with Vmaxof 7.11 nmol/mg protein/min. The low-affinity Km was 6.95 mmol, with Vmaxof 31.82 nmol/mg protein/min. In the cytosol there was a single Km of 0.30 mmol and Vmaxof 5.95 nmol/mg protein/min. In paired experiments examining differences between 1-d-old and adult rat lung, significantly higher total and mitochondrial malic enzyme activity-occurred in the newborn as compared with the adult. Malic enzyme activity was also present in the L2 cells. The finding of malic enzyme activity in the lung suggests that cytosolic malic enzyme may play a role in generating NADPH needed in the lung for fatty acid synthesis. These findings of developmental differences in malic enzyme activity suggest that alternate substrates such as anaplerotic amino acids may be used in the young animal as energy substrates by way of the tricarboxylic acid cycle.