The mechanism by which cells develop thermotolerance is unknown but some previous results suggest that membranes are involved in the process. The lipid composition of membranes from Ehrlich ascites cells grown in tissue culture was analysed at times up to 24 hours after heat treatments at 42 to 44 degrees C known to induce thermotolerance. No changes were observed in the levels of free cholesterol and phospholipids in the cells, nor in the fatty acid composition of the phospholipids. In addition, no changes were observed in the level of cholesterol esters. When cells were fractionated into crude nuclear, mitochondrial and microsomal fractions by differential centrifugation, there was still no heat-induced change in the free cholesterol or phospholipid levels of these fractions. It is concluded that thermotolerance is not mediated through a compositional change in the membranes to a more thermostable form.