Ethanol is known to alter the biochemical and biophysical properties of cellular membranes. In this investigation the mechanisms of ethanol-induced membrane potential changes in rat thymocytes were studied with fluorescence spectrophotometry using membrane potential-sensitive carbocyanine dyes. We analyzed (1) an ethanol-induced fluorescence increase (depolarization) in K-free saline-adapted cells, (2) a prolonged hyperpolarizing potential (fluorescence decrease) induced by adding small amounts of KC1 (1-5 mM) in K-+-free saline-adapted cells, and (3) a hyperpolarizing potential induced by the application of the Ca ionophore A23187 in normal saline. The ethanol-induced fluorescence increase in K-free saline solution was completely suppressed by the addition of a small amount of KC1 or the K ionophore valinomycin. Hyperpolarizing potentials induced by adding KC1 (1-15 mM) in K-free adapted cells were abolished by ouabain; this hyperpolarizing potential was not observed in the absence of Na in the bathing medium. These data indicate that the thymocytes possess a ouabain-sensitive electrogenic Na pump. The in vitro treatment of thymocytes with ethanol (200 mM) for up to 5 hours did not significantly alter this pump potential. However, in thymocytes derived from both ethanol-dependent intoxicated and ethanol-dependent withdrawing rats, the Na pump potential was significantly smaller than that observed in controls. The Ca-dependent hyperpolarization in response to A23187 (50 mM) was not significantly altered by the in vitro treatment of the cells with ethanol (200 mM) for 3 hours. These results suggest that ethanol can depolarize the membrane when the Na pump is inhibited by K-free saline and that alterations in Na pump activity may be associated with ethanol dependence.