In order to gain a better understanding of the molecular mechanism of the attenuated heat shock response in aging diploid fibroblasts (Liu, A. Y.-C., Lin, Z., Choi, H.-S., Sorhage, F., and Li, B. (1989) J. Biol. Chem. 264, 12037-12045), we examined the regulation of a heat-inducible, heat shock element (HSE) sequence-specific binding protein in IMR-90 diploid fibroblasts. Using gel retardation assay, we showed that the HSE binding activity in extracts of IMR-90 cells was very dependent on heat shock of the cells; that the induction was transient with a maximal increase observed at 1 h of heat shock. Significantly, the level of this heat-inducible HSE-binding activity was age-dependent, being high in young cells and low in old cells. By Scatchard analysis, we determined that this difference in HSE binding in young and old cells was not due to a change in the affinity of the binding, rather the level of the heat-inducible HSE-binding activity was different. The equilibrium dissociation constants (Kd) were estimated to be 30, 25, and 31 pM for cells with population-doubling levels of 22, 35, and 45, respectively; the corresponding Bmax values were 0.087, 0.042, and 0.0059 pmol of 32P-HSE bound per mg of whole cell extract protein. The decreased 32P-HSE-binding activity in the old cells was not a reflection of global alteration of many transcription factors; assay of the DNA binding activity of activating transcription factor showed little difference as a function of age. Experiments of mixing extracts from young and old cells provided evidence of a dominant inhibitor of the HSE-binding activity in old cells. Probing of protein blots with 32P-HSE showed that the subunit molecular weight of the HSE-binding protein was 83,000 in both young and old cells. The pattern of regulation of this HSE-binding protein upon heat shock and cell aging as well as the identity, in DNA sequence specificity and subunit molecular weight, of this protein with that of the human heat shock gene transcription factor suggest that the HSE-binding protein is involved in the transcriptional activation of hsp genes in IMR-90 cells. We concluded that there was an age-associated decrease in the heat shock gene transcription factor DNA-binding activity and that this could account for the attenuated heat shock gene expression in aging diploid cells.