The effects of N-methyl-D-aspartate (NMDA) on the generation of intracellular reactive oxygen species (ROS) and intracellular calcium in rat dissociated cerebellar cells were examined by flow cytometry. Flow cytometry allows the selection of a specific viable neuronal population with high sensitivity. We used 2',7'-dichlorofluorescin diacetate (DCFH-DA) as a marker of intracellular oxidative stress, and intracellular calcium was measured using Indo-1 as a calcium-sensitive indicator. The cerebellar cell population was isolated by size, granularity and NMDA-sensitivity by cell-sorting. In this cerebellar cell preparation, in which no glial cells were found, NMDA induced a concentration-dependent increase in ROS and intracellular calcium levels. These effects were inhibited by the non-competitive NMDA antagonist (+)MK-801. These results indicate that flow cytometry could be a useful tool to study the effect of neuroprotective drugs on NMDA receptor in isolated cerebellar neurons. Moreover, due to its high speed of analysis and the possibility to detect simultaneously a variety of fluorescent markers, we stated the utility of this technique in the pharmacology and physiology of the central nervous system.