L-Glutamine is a physiological inhibitor of endothelial NO synthesis. The present study was conducted to test the hypothesis that metabolism of glutamine to glucosamine is necessary for glutamine inhibition of endothelial NO generation. Bovine venular endothelial cells were cultured for 24h in the presence of 0, 0.1, 0.5 or 2mM D-glucosamine, or of 0.2 or 2mM L-glutamine with or without 20µM 6-diazo-5-oxo-L-norleucine (DON) or with 100µM azaserine. Both DON and azaserine are inhibitors of L-glutamine:D-fructose-6-phosphate transaminase (isomerizing) (EC 18.104.22.168), the first and rate controlling enzyme in glucosamine synthesis. Glucosamine at 0.1, 0.5 and 2mM decreased NO production by 34, 45 and 56% respectively compared with controls where glucosamine was lacking. DON (20µM) and azaserine (100µM) blocked glucosamine synthesis and prevented the inhibition of NO generation by glutamine. Neither glutamine nor glucosamine had an effect on NO synthase (NOS) activity, arginine transport or cellular tetrahydrobiopterin and Ca2+ levels. However, both glutamine and glucosamine inhibited pentose cycle activity and decreased cellular NADPH concentrations; these effects of glutamine were abolished by DON or azaserine. Restoration of cellular NADPH levels by the addition of 1mM citrate also prevented the inhibiting effect of glutamine or glucosamine on NO synthesis. A further increase in cellular NADPH levels by the addition of 5mM citrate resulted in greater production of NO. Collectively, our results demonstrate that the metabolism of glutamine to glucosamine is necessary for the inhibition of endothelial NO generation by glutamine. Glucosamine reduces the cellular availability of NADPH (an essential cofactor for NOS) by inhibiting pentose cycle activity, and this may be a metabolic basis for the inhibition of endothelial NO synthesis by glucosamine.