The relationship among membrane fatty acid composition, membrane fluidity, and stress tolerance was investigated in yeast cells. Several strains were examined for their ability to survive heat, ethanol, and hydrogen peroxide stresses. Membrane fluidity was determined by measuring fluorescence anisotropy using diphenylhexatriene as a probe. There was no obvious relationship among membrane fatty acyl composition, membrane fluidity, and stress tolerance in the strains examined. A consistent trend in the present study was an observed decrease in membrane fluidity following thermal treatment, which coincided with a reduction in cell viability. We suggest that protein denaturation may be responsible for the observed effect of elevated temperature on membrane fluidity and viability. This was implied by observations on the irreversible nature of thermal transitions, as measured by breaks in Arrhenius plots, in which stationary phase cells were shown to exhibit higher transition temperatures (53.9–55.5 °C) than exponential phase cells (49.5–51 °C). Furthermore, the thermal transition temperature was shown to increase in exponential phase cells following heat shock, which was associated with an increase in thermotolerance. We suggest that the thermotolerant state of heat-shocked cells and cells entering stationary phase may be associated with increased protein stability. However, despite the relatively good correlation between thermal transition temperature and stress tolerance, the thermal transition temperature did not predict the stress tolerance of a given strain, as stress-sensitive strains had similar transition temperatures to those of stress-resistant strains.Key words: membrane fluidity, stress tolerance, yeast, membrane lipids.