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Cyclosporine A (CsA) is the immunosuppressor most frequently used in transplant surgery and in the treatment of autoimmune diseases because of its specific inhibiting effect on signal transduction pathways of cell T receptor. It has been shown that CsA is able to generate reactive oxygen species and lipid peroxidation, which are directly involved in the CsA hepatotoxicity. In the present study, we investigated the effect of a sublethal heat pre-treatment (43°C for 30 min) on the hepatoma cell line HepG2 exposed to cytotoxic concentrations of CsA (10 and 25 μM) for 3 and 24 h. Parameters of cytotoxicity were assayed by measuring LDH (lactate dehydrogenase) leakage into the medium. Peroxide concentration was tested by flow cytometry by measuring the fluorescence intensity of DCF (dichlorofluorescein). Gene expression of catalase was detected by measuring the respective mRNA and proteins, as well as protein level of HSP70. The enzymatic activity of catalase was also determined. Heat pre-treatment significantly reduced CsA cytotoxicity as well as the level of peroxide generation. The protective effect of the previous heat treatment (corroborated by the irreversible catalase inhibitor 3-aminotriazole) against the CsA cytotoxicity was due to an increased expression and activity of catalase that was significantly reduced by the effect of CsA. We conclude that heat pre-treatment strongly protects against CsA injury, and the mechanism of this protection is by means of inducing not only the expression of HSP70 but also the expression and activity of catalase, the main enzyme system involved in H2O2 elimination.