We studied the mechanisms involved in heat gradient-induced thermotolerance of Saccharomyces cerevisiae. Yeasts were slowly heated in a nutrient medium from 25 to 50 degrees C at 0.5 degrees C/min or immediately heat shocked at 50 degrees C, and both sets of cultures were maintained at this temperature for 1 h. Cells that had been slowly heated showed a 50-fold higher survival rate than the rapidly heated cells. Such thermotolerance was found not to be related to protein synthesis. Indeed Hsp104 a known protein involved in yeast thermal resistance induced by a preconditioning mild heat treatment, was not synthesized and cycloheximide addition, a protein synthesis inhibitor, did not affect the thermoprotective effect. Moreover, a rapid cooling from 50 to 25 degrees C applied immediately after the heat slope treatment inhibited the mechanisms involved in thermotolerance. Such observations lead us to conclude that heat gradient-induced thermal resistance is not directly linked to mechanisms involving intracellular molecules synthesis or activity such as proteins (Hsps, enzymes) or osmolytes (trehalose). Other factors such as plasma membrane phospholipid denaturation could be involved in this phenomenon.