Induction of heat shock proteins (HSP) is generally regarded as a consequence of binding of the heat shock transcription factor (HSF) to heat shock elements (HSE), i.e. to be a single hit induction. The activation of HSF and the induction of HSP68 mRNA were studied in non pretreated Reuber H35 rat hepatoma cells in a thermosensitized and in a thermotolerant state. It was found that HSF in Reuber H35 hepatoma cells already acquires maximum DNA binding activity at temperatures that are too low to induce HSP68 mRNA. Directly following heat shock cells are in a transient thermosensitized state. In this state a second stress of lower impact leads to even higher production of HSP68, which corresponds with a decreased decay rate HSF-HSE binding. Directly following the thermosensitized state cells become refractory. In this period a second stress of the same impact does lead to HSF-HSE binding but the production of HSP68 mRNA is lowered, while only higher-impact stresses lead to high inductions of the said mRNA. The results indicate that regulation of HSP68 gene transcription involves at least one additional event outside the acquisition of DNA-binding activity by HSF and that this process can thus be described as a multiple-hit occurrence.