Analysis of the heat shock element (HSE)-binding proteins in extracts of rodent cells, during heat shock and their post-heat shock recovery, indicates that the regulation of heat shock response involves a constitutive HSE-binding factor (CHBF), in addition to the heat-inducible heat shock factor HSF1. We purified the CHBF to apparent homogeneity from HeLa cells using column chromatographic techniques including an HSE oligonucleotide affinity column. The purified CHBF consists of two polypeptides with apparent molecular masses of 70 and 86 kDa. Immunoblot and gel mobility shift analysis verify that CHBF is identical or closely related to the Ku autoantigen. The DNA binding characteristics of CHBF to double-stranded or single-stranded DNA are similar to that of Ku autoantigen. In gel mobility shift analysis using purified CHBF and recombinant human HSF1, CHBF competes with HSF1 for the binding of DNA sequences containing HSEs in vitro. Furthermore, when Rat-1 cells were co-transfected with human Ku expression vectors and the hsp70-promoter-driven luciferase reporter gene, thermal induction of luciferase is significantly suppressed relative to cells transfected with only the hsp70-luciferase construct. These data suggest a role of CHBF (or Ku protein) in the regulation of heat response in vivo.