In this study, to better understand the molecular mechanism underlying cellular responses to mild hyperthermia, we investigated gene expression patterns and genetic networks in human myelomonocytic lymphoma U937 cells using high-density oligonucleotide microarrays and computational gene expression analysis tools. The cells were incubated at 41 degrees C for 30 min (mild hyperthermia treatment) and then at 37 degrees C for 0-6 h. Although the mild hyperthermia treatment of the cells did not induce apoptosis, significant increases in the protein expression levels of heat shock proteins (HSPs), namely, Hsp27, Hsp40 and Hsp70, were observed following the activation of heat shock factor-1. Of the 22,283 probe sets analyzed, 423 probe sets were up-regulated and 515 probe sets were down-regulated by >1.5-fold in the cells 3 h post-treatment. Computational gene network analysis demonstrated that the significant genetic network A that contained many HSPs such as DNAJB1, HSPA1A, and HSPA1B was associated with cellular function and maintenance, post-transcriptional modification, or protein folding. Moreover, the significant genetic network B whose core contained v-myc myelocytomatosis viral oncogene homolog (MYC) was associated with cell morphology, cell cycle, and cellular development. The expression levels of nine selected genes were comparable to those determined by microarray analysis with real-time quantitative PCR assay. The present results indicate that mild hyperthermia affects the expression of a large number of genes and provides additional novel insights into the molecular basis of mild hyperthermia in cells.