The heat-shock response is characterized by the activation of heat-shock transcription factor 1 (HSF1), followed by increased expression of heat-shock proteins (Hsps). The stress-induced subnuclear compartmentalization of HSF1 into nuclear stress granules has been suggested to be an important control step in the regulation of stress response and cellular homeostasis in human cells. In this study, we demonstrate that the less-well characterized HSF2 interacts physically with HSF1 and is a novel stress-responsive component of the stress granules. Based on analysis of our deletion mutants, HSF2 influences to the localization of HSF1 in stress granules. Moreover, our results indicate that the stress granules are dynamic structures and suggest that they might be regulated in an Hsp70-dependent manner. The reversible localization of Hsp70 in the nucleoli strictly coincides with the presence of HSF1 in stress granules and is dramatically suppressed in thermotolerant cells. We propose that the regulated subcellular distribution of Hsp70 is an important regulatory mechanism of HSF1-mediated heat shock response.