Exposure of Listeria innocua to acid and starvation stress decreases sensitivity to the quaternary ammonium compound cetrimide, whereas exposure to cold and heat stress increases sensitivity to this compound. Changes in membrane lipids occur in response to certain types of stress, and these changes likely impact cell sensitivity to chemical sanitizers. The present study included an assessment of the effects of acid, starvation, cold, and heat stress on net cell hydrophobicity and fatty acid composition in L. innocua. Net cell hydrophobicity was determined by measuring absorbance of stress-adapted cell suspensions after partitioning with the nonpolar solvent n-hexadecane. Free fatty acids extracted from stress-adapted suspensions were analyzed by gas chromatography. Adaptation to acid and starvation increased net cell hydrophobicity and decreased membrane fluidity, which was correlated with reductions in anteiso fatty acids and in ratios of anteiso to iso fatty acids. Conversely, cold-stressed populations exhibited decreased net cell hydrophobicity and increased membrane fluidity with a corresponding increase in C15:C17 and anteiso:iso ratios and in C18 unsaturated fatty acids. No significant changes in net cell hydrophobicity or membrane fluidity were observed in heat-stressed cells, which exhibited increased sensitivity to cetrimide, suggesting another mechanism for altered cell sensitivity. These findings indicate that the efficacy of cetrimide against Listeria is partially dependent on the physiological state of the organism following exposure to various environmental stresses.