The inhibition of growth and cell wall synthesis by 3-amino-3-deoxy-D-glucose (3-AG), which is known to be one of the constituents of the kanamycin molecule and a metabolite of Bacillus sp., was almost completely overcome by glucosamine and N-acetylglucosamine in Staphylococcus aureus but scarcely affected by D-glucose and D-fructose. The antibiotic did not inhibit the incorporation of [14C]glucosamine and [3H]N-acetylglucosamine into the acid-insoluble fraction, but rather enhanced the incorporation of [14C]glucosamine. On the other hand, it inhibited the incorporation of D-[14C]fructose into the cell wall fraction but hardly affected the incorporation of D-[14C]fructose into the acid-insoluble fraction in the presence of pencillin G. Based on these results, it is suggested that the site of primary action of 3-AG is the formation of glucosamine-6-phosphate from D-fructose-6-phosphate, which is catalyzed by glucosamine synthetase [EC 2.6.1.16].