Endothelial cell dysfunction following exposure to H 2O 2 is associated with rapid inhibition of glucose-dependent pathways of ATP synthesis. The role other substrates for ATP synthesis (e.g., amino acids) may play in the metabolism of H 2O 2-injured cells is unclear. The effect of glutamine, a precursor of the Kreb's cycle intermediate α-ketoglutarate on ATP levels in bovine pulmonary artery endothelial cells exposed to H 2O 2 was examined. The presence of glutamine during H 2O 2 injury significantly enhanced ATP levels in the injured cells. Concentrations of glutamine as low as 50 μ M produced significant improvement of ATP levels in endothelial cells exposed to 5 m M H 2O 2. The 2 m M concentration of glutamine produced the greatest benefit, while greater concentrations of glutamine (5–20 m M) were actually associated with progressive decrements of the maximal benefit seen with the 2 m M concentration. The 2 m M concentration of glutamine produced similar enhancement of ATP with 1 and 10 m M H 2O 2 injury as well. Short-term viability following 5 m M H 2O 2 injury was significantly improved by the presence of 2 m M glutamine. The most effective concentration of glutamine (2 m M) did not scavenge H 2O 2 in a fluorometric assay. These observations suggest that mitochondrial substrates, such as glutamine, that bypass glucose-dependent pathways of ATP synthesis may be useful therapeutic agents for maintenance of ATP levels in oxidant-injured cells.