We have shown previously that human epidermal keratinocytes in situ and in vitro constitutively express high levels of the 72-kD heat shock protein (hsp72) and that hsp72 expression in these cells can be further induced with heat treatment. In the present study, we continue our investigation of the ultraviolet (UV) B protective effect of hyperthermic treatment and ask whether hsp72 is a mediator of heat-shock-induced UVB resistance. The results of our experiments demonstrate that heat treatment (42 degrees C for 4 h) before UVB exposure is able to increase significantly the UVB resistance of the epidermal carcinoma cell line A431. Heat-induced UVB resistance was most pronounced if the cells were exposed to UVB immediately after heat treatment. The protective effect was not detectable beyond a recovery period of 12 h. To investigate the role of hsp72 in hyperthermia-induced UVB resistance, we inhibited the expression of this protein using either a specific antisense oligodeoxynucleotide or quercetin, a flavonoid that has been shown to down-regulate hsp expression. Treatment with the oligomer as well as with quercetin significantly increased the susceptibility of A431 to UVB-induced damage and nullified the protective effect of heat preconditioning. A noncomplementary control oligodeoxynucleotide had no significant effect. These results indicate that heat treatment is able to induce a state of increased resistance to the deleterious effects of UVB in human keratinocytes in vitro. hsp72 is a molecular mediator of this protective effect, and its constitutive expression in human epidermal keratinocytes may be an important mechanism for the protection of human epidermis from UVB-induced damage.