Different requirements for glutamine of rats fed two amino acid diets were investigated in two rat-feeding experiments. In the first, the nonessential and essential amino acid mixtures were interchanged and the nonessential mixture was fed at 3 levels to determine whether the need for glutamine was related to the essential amino acids, the nonessential amino acids, or to their relative proportions. Results showed only the composition of the nonessential mixtures to be important. Because the mixtures differed primarily in their proportions of glycine and serine, these amino acids were tested in the second experiment by feeding 3 levels of each in all combinations, both with and without glutamine. Multiple regression analysis of the gains showed that additions of glycine or serine in excess of an undefined optimal level caused a linear reduction in gain and that the effects of each were additive. Inclusion of glutamine greatly reduced the inhibitory effects. Urine samples from selected groups were examined with an automatic amino acid analyzer. Increased excretion of a number of compounds resulted from feeding glycine and serine, which was prevented or greatly reduced by glutamine. These included, besides glycine and serine themselves, threonine, histidine, glutamine/asparagine, taurine, alanine, and lysine. The action of glutamine was accompanied by an increase in the excretion of urea and ammonia.