The fluorescent glucose analog, 2-(N-(7-nitrobenz-2-oxa-1,3diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), was used to measure rates of glucose uptake by single Escherichia coli cells. When cell populations were exposed to the glucose analog, 2-NBDG was actively transported and accumulated in single cells to a steadystate level that depended upon its extracellular concentration, the glucose transport capacity of the cells, and the intracellular degradation rate. The dependence upon substrate concentration could be described according to Michaelis Menten kinetics with apparent saturation constant K M =1.75 +M, and maximum 2-NBDG uptake rate=197 moleculesΓ cell-second. Specificity of glucose transporters to the analog was confirmed by inhibition of uptake of 2-NBDG by d-glucose, 3-o-methyl glucose, and d-glucosamine, and lack of inhibition by l-glucose. Inhibition of 2-NBDG uptake by d-glucose was competitive in nature. The assay for 2-NBDG uptake is extremely sensitive such that the presence of even trace amounts of d-glucose in the culture medium (t0.2 +M) is detectable. The rates of single-cell analog uptake were found to increase proportionally with cell size as measured by microscopy or single-cell light scattering intensity. The assay was used to identify and isolate mutant cells with altered glucose uptake characteristics. A mathematical model was developed to provide a theoretical basis for estimating single-cell glucose uptake rates from single-cell 2-NBDG uptake rates. The assay provides a novel means of estimating the instantaneous rates of nutrient depletion in the growth environment during a batch cultivation.