In Escherichia coli about one half of the negative supercoiling of DNA is constrained by proteins, in contrast to the situation in eukaryotic cells where most of the DNA is constrained by histones. The level of supercoiling in the unrestrained portion is controlled by a balance between the supercoiling activity of gyrase and the relaxing activity of DNA topoisomerase I. In the present work we show, by disrupting one or both genes encoding the heterodimeric protein HU, that an interplay exists in bacteria between HU and topoisomerase I activity: a decrease in the intracellular concentration of HU was accompanied by an increase in relaxing activity as measured in cell extracts. Conversely, a topA10 mutant of topoisomerase I, which has low levels of relaxing activity, was unable to accept an HU deficiency introduced by transduction. Thus it appears that the ability to increase relaxing activity, or to decrease an excess of supercoiling, is important for cells to survive in the absence of HU. These data can be explained in terms of HU constraining supercoiling in vivo as it does in vitro: the absence of HU would generate more unconstrained supercoiling, which in turn would require an increase in relaxing activity to maintain physiological levels.