L-Glutamine (0.02-2 mM) but not D-glutamine (0.2 mM and 2 mM) inhibited the release of endothelium-derived relaxing factor (EDRF) from bovine aortic endothelial cells cultured in the presence or absence of L-arginine. Inhibition was maximal at a concentration of 200 microns, and was reversed by L-arginine (50 microns) but not D-arginine (100 microns). L-Glutamic acid (2 mM) or ammonium chloride (1 mM), putative products of the metabolism of L-glutamine in endothelial cells, had no effect on EDRF release. L-Glutamine (0.2 mM and 2 mM) but not D-glutamine (2 mM), L-glutamic acid (2 mM) or ammonium chloride (1 mM) also inhibited the generation of L-arginine in endothelial cells. Thus, L-glutamine inhibits EDRF release by preventing the generation of L-arginine.