A mutant was isolated from Escherichia coli K-12 which requires glucosamine or N-acetylglucosamine for growth. Depriving the mutant of glucosamine resulted in a rapid loss of viability of the cells, followed by a decrease in the turbidity of the culture. When the mutant cells were resuspended in broth media containing 10% sucrose, the rod-shaped cells became spheroplasts. However, the presence of sucrose in the media did not prevent the cells from losing their viability. This mutant was shown to be deficient in the activity of l-glutamine:d-fructose-6-phosphate aminotransferase (EC 2.6.1.16). The activity of the deaminating enzyme, 2-amino-2-deoxy-d-glucose-6-phosphate ketol-isomerase (EC 5.3.1.10), appeared to be normal in this mutant. The position of the mutation has been determined to be at the 74th min of the Taylor and Trotter map, as shown by cotransduction with phoS (90%) and ilv (25%) by using bacteriophage P1.