Neurons and glia of mature brain can be distinguished by their isoenzyme content of the glycolytic enzyme enolase. Neurons contain neuron-specific enolase (NSE) and glial cells have non-neuronal enolase (NNE). Measurement of each isoenzyme by specific radioimmunoassay during the course of brain development in rat shows that NSE levels are very low in embryonic brain and increase at a time coincident with the morphological and functional maturation of neurons. NNE levels are high in embryonic brain and decrease when NSE first appears, followed by a gradual increase to adult levels. NSE levels rise at a slower rate in brain areas known to develop over a more protracted period (forebrain, cerebellum) compared to areas that develop more rapidly (brain stem). The data are consistent with a hypothesized switch from NNE to NSE during neuronal development. In E60 and E100 monkey brain tissue NSE/NNE ratios are higher in regions containing older neurons. This suggests that a similar switch from NNE to NSE also occurs during neuronal development in monkey.