Genetic studies revealed that DNA gyrase seems to catalyze immediate and transient DNA relaxation after Escherichia coli cells are exposed to heat shock (Ogata, Y., Mizushima, T., Kataoka, K., Miki, T., and Sekimizu, K. (1994) Mol. Gen. Genet. 244, 451-455). We have now obtained biochemical evidence to support this hypothesis. DNA gyrase catalyzed an increase in the linking number of DNA and relaxation of negatively supercoiled DNA, under physiological concentrations of ATP. Analyses by gel filtration chromatography of each subunit revealed that DNA relaxation activity co-migrated with each subunit. The linking number of DNA increased as the temperature increased. Further, the reaction was inhibited by nalidixic acid or by oxolinic acid. Based on these results, we propose that DNA gyrase participates in a concerted reaction with DNA topoisomerases in the immediate relaxation of DNA in cells exposed to heat shock.