Transcriptional induction of the hsp70 gene is mediated by heat shock factor 1 (HSF1) rapidly activated upon heat and other stresses. HSF2 has been thought to be responsible for accumulation of HSP70 during hemin-induced differentiation of human K562 erythroleukemia cells because of accompanying acquisition of HSF2 DNA binding activity. However, there has not been any direct evidence for such a functional role of HSF2. The purpose of this study is to clarify the roles of HSF1 and HSF2 in HSP70 induction in hemin-treated K562 cells. We show here that a chimeric polypeptide of HSF2 and GAL4 DNA binding domain (GAL4-BD-HSF2) was unable to induce a GAL4 binding site-containing luciferase reporter gene in response to hemin and that exogenously overproduced HSF2 also failed to increase expression of a heat shock element-containing reporter. On the contrary, expression of a GAL4-BD-HSF1 chimeric protein responded to hemin treatment as well as to heat shock, and transiently overexpressed HSF1 caused hemin-responsive induction of the reporter gene in a dose-dependent manner. These results indicate that HSF1, rather than HSF2, primarily mediates the hemin-induced transcription of the hsp70 gene.