Temperature shift is often practiced in the cultivation of Escherichia coli to reduce undesired metabolite formation and to maximize synthesis of correctly folded heterologous protein. As the culture temperature is decreased below the optimal 37°C, growth rate decreases and many physiological changes occur. In this study, we investigated the gene expression dynamics of E. coli on switching its cultivation temperature from 37 to 33 and 28°C using whole genome DNA microarrays. Approximately 9% of the genome altered expression level on temperature shift. Overall, the alteration of transcription upon the downshift of temperature is rapid and globally distributed over a wide range of gene classes. The general trends of transcriptional changes at 28 and 33°C were similar. The largest functional class among the differentially expressed genes was energy metabolism. About 12% of genes in energy metabolism show a decrease in their level of expression, and ∼6% show an increase. Consistent with the decrease in the glucose uptake rate, many genes involved in glycolysis and the PTS sugar transport systems show decreased expression. Genes encoding enzymes related to amino acid biosynthesis and transport also have reduced expression levels. Such decrease in expression probably reflects the reduced growth rate and the accompanying reduction in energy and amino acid demand at lower temperatures. However, nearly all genes encoding enzymes in the TCA cycle have increased expression levels, which may well be compensating the reduction of the activity of TCA cycle enzymes at lower temperatures. Temperature shift also results in shift of the cytochromes from the high affinity cytochrome o system to the low affinity cytochrome d system. There is no evidence that protein processing genes are selectively altered to create favorable conditions for heterologous protein synthesis. Our results indicate that the beneficial effect of temperature shift in many biotechnological processes is likely to be attributed to the general effect of reduced growth and metabolism.