Prior work employing an in vitro model of the cerebral cortex has shown that sodium pump activity is a critical determinant for neuronal survival of glutamate stimulation. We have hypothesized that up-regulation of total brain sodium pump activity will protect against potential excitotoxins. Increased sodium pump activity could theoretically occur by changes in the reaction rate (short-term) and/or by increased levels of sodium pump protein (long-term) and is potentially complex since the three catalytic (a) subunit isoforms of the sodium pump are distributed in a highly variable, cell-specific pattern in the brain. Short-term regulation (seconds to minutes) has been well studied: brain sodium pump exhibits a large dynamic range. In contrast, the possibility of long-term modulation of sodium pump activity has not been extensively explored. We used isoform specific antibodies and [H-3]ouabain binding to determine whether prolonged stimulation of sodium pump activity in rodent telencephalic cultures increased total sodium pump enzyme. Exposure of mixed neuronal-glial cultures to high levels of glutamate (10 mM) for 18 h, which is highly toxic to neurons, was associated with an approximate to 80% increase in alpha 1 and alpha 2 subunit expression by glia. Induction of alpha 2 subunit immunoreactivity was also associated with comparable changes in [H-3]ouabain binding, suggesting that the up-regulation corresponded to functional alpha 2 protein. Shorter (30 min) glutamate treatments, which also killed neurons, did not produce similar changes in sodium pump expression. In contrast to mixed cultures, pure astrocyte cultures had undetectable alpha 2 and alpha 3 and moderate levels of alpha 1 protein, as confirmed by low levels of [H-3]ouabain binding. Glutamate treatment using this protocol was associated with a decrease in alpha 1 sodium pump expression. We conclude that long-term regulation of the sodium pump can be demonstrated in glia which have developed in the presence of neurons. Both alpha 1 and alpha 2 isoforms of the sodium pump are involved in this response to glutamate.