Various parameters of anion and cation transport were measured in the cerebral cortex of neonatal (3-day-old) and adult rats following acute and chronic treatment with phenytoin (PHT). Acutely, PHT significantly inhibited the enzyme Na+, K+-ATPase in both neonatal and adult rats. This effect was accompanied by a significant increase in cerebral cortical Na+ content and a decrease in K+ content only in neonatal animals. Chronic treatment (two and four times a day for 7 days) of adult rats with PHT significantly reduced Na+ content without affecting whole homogenate Na+, K+-ATPase activity. The activity of this enzyme was markedly increased in the myelin- (glial product) and slightly decreased in the synaptosomal- (neuronal) fractions following chronic (four times a day for 7 days) PHT treatment. These results suggest that PHT differentially affects the two forms (neuronal and glial) of the enzyme Na+, K+-ATPase. The possible relevance of this hypothesis in relationship to the anticonvulsant and excitatory properties of PHT is discussed. Chronic (two and four times a day for 7 days) PHT treatment increased both DNA content and activity of the glial marker enzyme carbonic anhydrase. Activity of the mitochondrial enzyme HCO3- -ATPase was also increased following chronic PHT treatment. These two enzymes are intimately involved in the regulation of HCO3- -Cl- transport across glial cell and mitochondrial membranes, and these results suggest that PHT is able to affect beneficially glial regulatory processes. The ability to enhance glial regulation of anions and cations in extracellular fluid provides new and important insights into the mechanism of the anticonvulsant action of PHT.