Saccharomyces cerevisiae HSc was grown with ethanol at concentrations up to 10% (v/v). The immediate effects of additions of externally added ethanol on CO2 production and O2 consumption of washed organisms were studied by stopped-flow membrane inlet quadrupole mass spectrometry. Fermentative activities of organisms grown with ethanol (0-5% v/v) showed similar sensitivities to inhibition by ethanol, whereas those grown with 10% (v/v) ethanol had become protected and were markedly less sensitive. The fluidity of subcellular membrane fractions was measured by determination of the temperature dependence of the rotational order parameter of the spin label 5-doxyl stearic acid (free radical) by electron spin resonance. Mitochondria prepared from yeasts grown with 0, 7, and 9% (v/v) ethanol showed similar overall fluidity, although differences in temperature-dependent behaviour indicate altered lipid composition or lateral phase separations. On the other hand the microsomal fraction from organisms grown with 9% ethanol showed a remarkable increase in fluidity. These data suggest that the protective effects of growth with ethanol near the limit of tolerance on fermentative activities may arise from altered plasma membrane fluidity properties.