The fluorescent, non-metabolizable glucose analog 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-6-deoxyglucose (NBDG) was used to measure rates of hexose transport by dissociated brain cells from developing and adult rats. Flow cytometric analysis of glucose uptake and expression of glucose transporters was performed by mapping on size by granularity, which discriminated between neurons and astrocytes in a suspension of mixed brain cells. These mapped cell populations were identified by immunofluorescent staining with antisera to neuron-specific enolase (NSE) and glial fibrillary acidic protein (GFAP). Specific uptake of the analog by membrane glucose transporters was confirmed by its inhibition by D-glucose and by cytochalasin B. Both neurons and astrocytes expressed the GLUT1 and GLUT3 transporter isoforms. This was confirmed by the additive inhibition of NBDG uptake by antibodies to these transporter isoforms in both cell types. The advantages of flow cytometric analysis of glucose transport include continuous monitoring over extremely short periods of time, increased precision of cell-by-cell flow cytometric measurements versus average uptake rates obtained with radioisotopes, and simultaneous analysis of uptake by different cell populations. Moreover, both uptake rates and the abundance of specific transporters can be determined directly and rapidly on the same cell suspension.