Plasmid DNA in exponentially growing Escherichia coli immediately relaxes after heat shock but the relaxed DNA re-supercoils rapidly, the despite continued presence of the heat shock conditions. We have now obtained genetic evidence indicating that the histone-like protein HU of E. coli is required for this re-supercoiling of DNA. Plasmid DNA in a hupA-hupB double gene-disruption mutant relaxed excessively after heat shock, while the relaxation of DNA in a himA-himD double gene-disruption mutant and in an hns insertion mutant was transient, thereby indicating that HU protein, but not IHF or H-NS proteins, is required for the re-supercoiling of DNA. Exposure of the hupA-hupB double mutant to a temperature of 50 degrees C led to both excessive relaxation of DNA and to a decrease in viable cell number but temperatures lower than 46 degrees C did not lead to these events. Based on these results, we propose that HU protein maintains the negative supercoiling of DNA during thermal stress and contributes to cellular thermotolerance in E. coli.