Both non-arginine-depleted and arginine-depleted pulmonary artery endothelial cells (PAEC) actively convert citrulline into arginine. Exposure to hypoxia for 4-24 h inhibited arginine synthesis from citrulline in intact cells and in cell homogenates. The conversion of L-citrulline to L-argininosuccinate by argininosuccinate synthetase (AS) was inhibited by exposure to hypoxia for 4, 12, or 24 h. The conversion of argininosuccinate to arginine by argininosuccinate lyase was inhibited by exposure to hypoxia for 24 h but not for 4-12 h. The decrease of L-arginine biosynthesis during hypoxia coincided with the increase of intracellular glutamine content and was abrogated by preventing an increase in intracellular glutamine. In addition, AS activity was inversely related to glutamine content in the medium. These results indicate that hypoxia inhibited the L-arginine biosynthetic pathway via decreased activity of AS. The latter is related to increased glutamine content. Hypoxic inhibition of arginine synthesis from citrulline did not result in a decrease of arginine content, suggesting that PAEC are able to maintain intracellular arginine for up to 24 h despite reduction in the L-arginine biosynthetic pathway.