Escherichia coli glucosamine-6-phosphate isomerase is specific for removal of the 1-pro-R hydrogen of fructose 6-phosphate (fructose-6-P). The conversion of [2-3H]glucosamine-6-P to fructose-6-P plus ammonia is accompanied by 99% exchange of tritium with water and 0.6% transfer to C-1 of fructose-6-P. The enzyme is active toward alpha-glucosamine-6-P and apparently inactive toward the beta anomer. The combination of the above results supports a cisenolamine intermediate for the reaction. The labeling of substrate and product pools in tritiated water shows that the two halves of the reaction are each freely reversible. No single step appears to be rate determining. 2-Amino-2-deoxyglucitol-6-P is an unusually strong competitive inhibitor (K1 = 2 X 10(-7) M, compared with the Km = 4 X 10(-4) M for glucosamine-6-P), suggesting the enzyme has a strong affinity for the open-chain form of glucosamine-6-P.