We hypothesized that broad-scale expression profiling would provide insight into the regulatory pathways that control gene expression in response to stress and potentially identify novel heat-responsive genes. HEp2 cells, a human malignant epithelial cell line, were heated at 37 degrees C to 43 degrees C for 60 min to gauge the heat shock response, using as a proxy inducible Hsp70 quantified by Western blot analysis. Based on these results, microarray experiments were conducted at 37 degrees C, 40 degrees C, 41 degrees C, 42 degrees C, and 43 degrees C. Using linear modeling, we compared the sets of microarrays at 40 degrees C, 41 degrees C, 42 degrees C, and 43 degrees C with the 37 degrees C baseline temperature and took the union of the genes exhibiting differential gene expression signal to create two sets of "heat shock response" genes, each set reflecting either increased or decreased RNA abundance. Leveraging human and mouse orthologous alignments, we used the two lists of coexpressed genes to predict transcription factor binding sites in silico, including those for heat shock factor (HSF) 1 and HSF2 transcription factors. We discovered HSF1 and HSF2 binding sites in 15 genes not previously associated with the heat shock response. We conclude that microarray experiments coupled with upstream promoter analysis can be used to identify novel genes that respond to heat shock. Additional experiments are required to validate these putative heat shock proteins and facilitate a deeper understanding of the mechanisms involved during the stress response.