A combination of three amino acids including 1.0 g/L isoleucine, 0.5 g/L methionine and 2.0 g/L phenylalanine was found to enhance ethanol tolerance of a self-flocculating fusant of Schizosaccharomyces pombe and Saccharomyces cerevisiae. When subjected to 20% (V/V) ethanol for 9 h at 30 degrees C, all cells died whereas 57% remained viable for the cells grown in the presence of the three amino acids. Based on the analysis of protein amino acid composition of plasma membranes and the determination of plasma membrane fluidity by measuring fluorescence anisotropy using diphenylhexatriene as a probe, it was found that the significantly increased ethanol tolerance of cells grown with the three amino acids was due to the incorporation of the supplementary amino acids into the plasma membranes, thus resulting in enhanced ability of the plasma membranes to efficiently counteract the fluidizing effect of ethanol when subjected to ethanol stress. This is the first time to report that plasma membrane fluidity can be influenced by protein amino acid composition of plasma membranes.