In synaptosomal preparations from rat cerebral cortex, ouabain-sensitive Rb+ uptake was stimulated by ethanol (20-80 mM). Based on differential sensitivity to ouabain, 80% of this Na+,K(+)-ATPase activity represented activity of the alpha 1 isozyme while 20% was due to the alpha 2 and/or alpha 3 isozymes (alpha 2/ alpha 3). Stimulation of Na+,K(+)-ATPase was selective for the activity of alpha 2/alpha 3 which was increased by 167% in the presence of 80 mM ethanol. In this concentration range, ethanol had no effect on alpha 1 activity. Exposure of synaptosomal preparations to EGTA increased basal (no ethanol) alpha 2/alpha 3 activity with no effect on alpha 1 activity. Further, ethanol no longer stimulated alpha 2/alpha 3 activity after EGTA treatment. An EGTA extract was concentrated and desalted to yield a fraction that selectively inhibited alpha 2/alpha 3 activity when reconstituted with EGTA-treated synaptosomal preparations. This inhibition was trypsin-sensitive, suggesting protein involvement, and was prevented by 80 mM ethanol. In the presence of the inhibitory protein fraction, ethanol stimulated Na+, K(+)-ATPase activity in EGTA-treated membranes with a dose-response like that observed with the crude (no EGTA) synaptosomes. We propose that the alpha 2/alpha 3 activity of Na+,K(+)-ATPase is subject to inhibitory regulation and that ethanol stimulates this activity by releasing it from inhibition, an effect that may mimic in vivo deregulation of the enzyme by ethanol.