Glutamine is an important mitochondrial substrate implicated in the protection of cells from oxidant injury, but the mechanisms of its action are incompletely understood. Human pulmonary epithelial-like (A549) cells were exposed to 95% O2 for 4 days in the absence and presence of glutamine. Cell proliferation in normoxia was dependent on glutamine, and glutamine deprivation markedly accelerated cell death in hyperoxia. Glutamine significantly increased cellular ATP levels in normoxia and prevented the loss of ATP in hyperoxia seen in glutamine-deprived cells. Mitochondrial membrane potential as assessed by flow cytometry with chloromethyltetramethylrosamine was increased by glutamine in hyperoxia-exposed A549 cells, and a glutamine dose-dependent increase in mitochondrial membrane potential was detected. Glutamine-supplemented, hyperoxia-exposed cells had a higher O2 consumption rate and GSH content. Electron and fluorescence microscopy revealed that, in hyperoxia, glutamine protected cellular structures, especially mitochondria, from damage. In hyperoxia, activity of the tricarboxylic acid cycle enzyme α-ketoglutarate dehydrogenase was partially protected by its indirect substrate, glutamine, indicating a mechanism of mitochondrial protection.