The human heat shock transcription factor (HSF) is maintained in an inactive non-DNA-binding form under nonstress conditions and acquires the ability to bind specifically to the heat shock promoter element in response to elevated temperatures or other conditions that disrupt protein structure. Here we show that constitutive overexpression of the major inducible heat shock protein, hsp7O, in transfected human cells reduces the extent of HSF activation after a heat stress. HSF activation was inhibited more strongly in clones that express higher levels of hsp7O. These results demonstrate that HSF activity is negatively regulated in vivo by hsp7O and suggest that the cell might sense elevated temperature as a decreased availability of hsp7O. HSF activation in response to treatment with sodium arsenite or the proline analog azetidine was also depressed in hsp7O-expressing cells relative to that in the nontransfected control cells. As well, the level of activated HSF decreased more rapidly in the hsp7O-expressing clones when the cells were heat shocked and returned to 37°C. These results suggest that hsp7O could play an active role in the conversion of HSF back to a conformation that does not bind the heat shock promoter element during the attenuation of the heat shock response.