Compartmentalization of brain ascorbate and glutathione between neurons and glia has been a source of controversy. To address this question, we determined the ascorbate and glutathione contents of brain tissue with defined, but varying, densities of neurons and glia. In developing rat cortex and hippocampus, glutathione content rose during gliogenesis, while ascorbate fell. By contrast, ascorbate, but not glutathione, increased markedly during granule cell proliferation and maturation in the developing cerebellum. Similarly, in tissue from adult cerebral cortex of species with distinct neuron densities, ascorbate content increased linearly with increasing neuron density in the order: human<rabbit<guinea-pig<rat<mouse, whereas glutathione was relatively constant. These data suggest that ascorbate predominates in neurons, whereas glutathione is slightly predominant in glia. Quantitative analysis of ascorbate and glutathione contents in these studies combined with appropriate intra- and extracellular volume fraction data permitted calculation of concentrations of ascorbate in neurons (10mM) and glia (0.9mM), and glutathione in neurons (2.5mM) and glia (3.8mM). The relative accuracy of these values was confirmed by their use in a model that reliably predicted changes in ascorbate and glutathione levels in rat cortex during the first three postnatal weeks and into adulthood. These findings not only provide new information about the intracellular composition of neurons and glia, but also have implications for understanding the roles of ascorbate and glutathione in normal brain function, as well as neuron and glia involvement in disease states linked to oxidative stress.