We have demonstrated a positive correlation between membrane microviscosity and the temperature required to kill E. coli. Batches of cells with differing unsaturated fatty acid (u.f.a.) compositions were prepared from the u.f.a.-requiring E. coli K12 mutant K1060. The membrane microviscosity of these cells is estimated from the extent of fluorescence polarization of the probe molecule 1,6-diphenyl-1,3-5,-hexatriene dissolved in the membrane. For the same growth temperature, cells grown in oleic acid (18:1) have a greater microviscosity and u.f.a. content than linolenic acid (18:3) grown cells. the rate of decrease in microviscosity with increasing temperature is correlated with the amount of u.f.a. present in the membrane. From survival curves determined at several hyperthermic exposures, one can interpolate the hyperthermic temperature required to kill 90 per cent of the cells in three hours. These equivalent kill temperatures are directly related to the cell microviscosity. These data support the hypothesis that cell membrane microviscosity plays a critical role in hyperthermic killing.