The ethanol tolerance of Clostridium thermosaccharolyticum HG8 has been studied in continuous culture using a new technique that requires knowledge of the kinetic constants and measurement of substrate concentrations at various concentrations of the inhibitor. Endogenously produced ethanol was supplemented with exogenously supplied ethanol to achieve various inhibitor concentrations. The ethanol tolerance of C. thermosaccharolyticum was significantly greater than expected on the basis of most previous reports, which may be explained in part by acclimation occurring over time periods exceeding those typical of batch systems studied previously. An ethanol concentration of 40 g/L is required for 50% growth inhibition of C. thermosaccharolyticum at 55 °C. Process considerations suggest that the ethanol tolerance of C. thermosaccharolyticum is unlikely to significantly constrain its use for ethanol production from cellulosic biomass. Ester-linked phospholipid membrane analyses (ELPFA) revealed that growth in the presence of high concentrations of ethanol (33 g/L) resulted in a membrane profile having increased fluidity and molecular diversity. Ethanol-induced changes included a significant increase in shorter chain unsaturated fatty acids (C15:0) at the expense of longer chain unsaturated fatty acids (C17:0) and a slight increase in the amount of mono-unsaturated fatty acids.