An increase in extracellular nucleotides appeared to conform to simple diffusion, when Saccharomyces cerevisiae cells were suspended in 20% ethanol at 15 degrees C. However, in the case of cells grown in the presence of 8% ethanol, the leakage of nucleotide appeared to be repressed significantly in the initial phase. The addition of glucose led to a continuation of the repression of the leakage. Under this condition, the addition of iodoacetamide as an inhibitor in glycolytic pathway, or stilbestrol as an inhibitor of plasma membrane ATPase, resulted in a rapid increase in the leakage of nucleotides, indicating that the membrane permeability barrier is dependent on membrane ATPase. Ouabain, an inhibitor of the Na+/K+-ATPase, had no effect. The addition of 10 mM CaCl2 for inducing lateral phase separation in the inner membrane, which caused a faster release of nucleotides in general, had a little effect on nucleotide leakage, when cells grown in the presence of 8% ethanol were suspended in 20% ethanol containing glucose. Moreover, the addition of 10 mM KCl, together with CaCl2, had almost no effect on leakage. Ca2+ influx was found to be smaller in cells grown in the presence of ethanol when compared to cells grown in the absence of ethanol, when fura-2 loaded cells were suspended in K-MOPS buffer containing 50 mM CaCl2. Divalent cations such as Ba2+, Mg2+ and Mn2+ had effects similar to Ca2+ on membrane permeability. The decrease in cell viability corresponded to the amount of leakage of nucleotides over the experimental time course. These results suggest that the high activity of membrane ATPase which is induced by growth in the presence of 8% ethanol ensures homeostasis of ions in the cytoplasm at high concentrations of ethanol, and reduces the effects of membrane surface-acting substances such as divalent cations on the membrane integrity. Thus the maintenance of the cell membrane as a permeability barrier appears to lead to a high ethanol-endurability.