Mammalian cells exhibit increased sensitivity to hyperthermic temperatures of 38-42 degrees C after an acute high-temperature heat shock; this phenomenon is known as thermo-sensitization or the step-down heating effect. In order to determine whether the increase of heat shock mRNA after heat stress can be thermosensitized, we studied the induction of the mRNA of HSP68 and of HSP84 after application of step-down heating (SDH) in Reuber H35 rat hepatoma cells. SDH consisted of a pretreatment of 30 min at 41.5, 42.5 or 43.5 degrees C, followed by a continuous incubation at a lower hyperthermic temperature (40 or 41 degrees C). After mild pretreatment (30 min at 41.5 degrees C) the mRNA level of HSP68 was increased by subsequent incubation at 40 degrees C, although incubation at 40 degrees C alone had no effect. This increase was even more pronounced at 41 degrees C. An increase in the level of HSP84 mRNA was also observed after mild pretreatment (41.5 degrees C/30 min) followed by 41 degrees C post-incubation. Interestingly, an enhanced occurrence of thermotolerance was also observed upon application of mild step-down heating (42 degrees C/30 min-40 degrees C-43.5 degrees C/60 min). In contrast, cell cultures treated for 30 min at 43.5 degrees C (a temperature which induces an increase in HSP mRNA levels) showed an inhibited or delayed synthesis of HSP mRNA when post-treated at 40 or 41 degrees C. Under these conditions the development of thermotolerance did not take place either. With respect to the effect of step-down heating on HSP mRNA levels as well as on thermotolerance development, our data imply that a distinction should be made between 'mild' and 'severe' pretreatment temperature of the step-down heating protocol.