The effects of chronic ethanol exposure on Na+, K(+)-ATPase were investigated in PC 12 cells. Inclusion of ethanol in the Na+, K(+)-ATPase assay (i.e. in vitro addition of ethanol) inhibited enzyme activity. Conversely, intrinsic Na+, K(+)-ATPase activity was increased after chronic ethanol exposure of the cells. This increase in Na+, K+ pumps occurred without any alteration in the inhibitory effects of in vitro ethanol. A similar response was observed when the chronic treatments were carried out using serum-free defined medium. The effects of other agents, which like ethanol decrease membrane order, were investigated. The addition of ketamine and tert-butanol in vitro caused a concentration-dependent inhibition of Na+, K(+)-ATPase activity. However, chronic exposure of the PC 12 cells to tert-butanol or ketamine did not alter either intrinsic Na+, K(+)-ATPase activity or the inhibitory effects of ethanol in vitro. Maintenance of PC 12 cells in medium containing ethanol resulted in an increase in the intracellular content of Na+ without any change in the K+ levels. In contrast, maintenance of the cells in medium containing tert-butanol did not alter intracellular levels of Na+ or K+. The present study shows that the ethanol-induced increase in Na+, K+ pumps involved an increase in the intracellular content of Na+. This increase in Na+ content did not appear to be secondary to an inhibition of Na+, K(+)-ATPase activity.