A fluorescent derivative of D-glucose, 2-NBDG, which was previously developed for the evaluation of glucose uptake activity by living cells, was used on Escherichia coli cells and its fate after incorporation in the cells was investigated. 2-NBDG was converted to another fluorescent derivative (2-NBDG metabolite) immediately after it was taken by E. coli cells. This 2-NBDG metabolite was then decomposed to non-fluorescent forms. 2-NBDG metabolite was decomposed into the original 2-NBDG by G6Pase with concurrent liberation of inorganic phosphate. Furthermore, FAB/MS analysis showed that its molecular weight was 420, the same value as that of 2-NBDG 6-phosphate. These indicate 2-NBDG metabolite should be 2-NBDG 6-phosphate. Based on these results, the feasibility of 2-NBDG as a fluorescent non-toxic probe for glucose uptake activity and its application to viability assessment of various living systems are discussed.