Background: The mechanisms by which moderate hypothermia (32°C for 12 - 72 hours) affect human cellular function are unclear. We tested the hypothesis that it produces broad changes in mRNA expression in vitro. Methods: Acute monocytic leukemia (THP-1) cells were incubated under control conditions (37°C) or moderate hypothermia (32°C) for 24 hours. RNA was extracted and a hypothermic response was confirmed by examining expression of the cold-inducible gene CIRBP by reverse transcription PCR. Gene expression analysis was performed on seven sets of paired samples with Affymetrix U133A chips, using established statistical methods. Sequences were considered affected by cold if they showed statistically significant changes in expression and also met published post-hoc filter criteria (changes in geometric mean expression of 2-fold or greater and expression calls of "present" or "marginal" in at least half of the experiments). Changes in expression of selected sequences were further confirmed by PCR. Results: 67 sequences met criteria for increased expression (including cold-inducible genes CIRBP and RBM3), and 100 sequences showed decreased expression as a result of hypothermia. Functional categories affected by hypothermia included genes involved in immune responses, cell growth, proliferation, and differentiation, and metabolism and biosynthesis. Several heat shock proteins (HSPs) showed decreases in expression. Conclusions: Moderate hypothermia produces substantial changes in gene expression, in categories potentially of systemic importance. Cold exposure without re-warming decreased expression of several HSPs. These in vitro findings suggest that prolonged hypothermia in vivo might be capable of producing physiologically relevant changes in gene expression by circulating leukocytes.