We have studied mechanisms of HSP70 gene regulation at 37 degreesC by the cellular factors NF-IL6 and Ku70. As both factors repress HSF1, we first examined whether phosphorylation on serine 303 and 307 of HSF1 by MAPK and GSK3, which has known to inhibit HSF1, was involved in the repression. However, repression by NF-IL6 was found using HSF1 mutants S303G and S307G refractory to the effects of MAPK and GSK3. We then examined whether NF-IL6 repressed HSP 70B by a mechanism resembling Ku proteins. However, in Ku-deficient cells, NF-IL6 was still able to displace HSF1 from heat shock element (HSE) and repressed HSF1 activation. In addition, activation of the HSP70B promoter by wild type, S303G, or S307G HSF1 was observed to be much more pronounced in Ku-deficient cells. In vitro translated Ku70 interacted with HSF1 by binding to and displacing it from HSE. These data indicate that the repression of the HSP70B promoter by NF-IL6, Ku70, and MAPK occurs independently of each other and involves three complementary mechanisms.