In order to use the Drosophila melanogaster S2 cell line in the manufacture of biotechnological products, the study of its metabolic aspects is of fundamental importance. The present work aimed to identify the effect of glutamine fed-batch on a D. melanogaster S2 stably transfected for synthesis of the rabies virus glycoprotein (rRVGP). For this purpose initial GLN concentration was reduced and a fed-batch strategy was used to supplement this amino-acid in suspension cultures carried out in 250 mL spinner flask at 80 rpm, 28°C and pH5.8. GLN initial concentration was reduced to 1.23g/L and 0.53g/L, and supplementation of GLN was performed during the cell growth. As result the ammonium formation decreased, and the supply of extra glutamine in fed-batch mode runs extended the exponential phase, but was not sufficient to revert the metabolic response to low glutamine.