We have studied the effect of altering the in vivo level of DNA supercoiling on the phase-variable expression of the Escherichia coli fimA gene. Transcription from the fimA promoter was unaffected by changes in DNA supercoiling whether caused by the introduction of a topA::Tn10 mutation or by inhibition of DNA gyrase with the antibiotic novobiocin. However, inversion of the fimA promoter fragment was altered in response to perturbation of DNA supercoiling. Specifically, inactivation of topA reduced the rate of promoter fragment inversion in both the ON-to-OFF and the OFF-to-ON directions. This effect correlated with the loss of functional topA and not with the global level of DNA supercoiling. Inhibition of DNA gyrase introduced a bias in favour of the OFF-to-ON inversion; the ON-to-OFF inversion was affected only slightly. Changes in expression of fimB, the gene coding for the recombinase that catalyses fimA promoter fragment inversion in the strains used in this study, did not correlate with effects on fimA phase variation: we found that transcription of fimB was inhibited by loss of functional topA and was enhanced by inhibition of DNA gyrase in a manner that correlated well with the global level of in vivo DNA supercoiling. A model is presented to account for the effects of lost topoisomerase function on fimA gene expression.