The effect of FtsH, an essential inner membrane-bound protease, in the regulation of the sigma 54-dependent Pu promoter has been examined in vivo. Escherichia coli cells lacking FtsH failed to activate a Pu-lacZ fusion in response to the cognate enhancer-binding protein XylR. However, the intracellular concentrations of XylR and sigma 54, as well as their apparent physical integrity were the same regardless of the presence or absence of the protease. The loss of Pu activity in FtsH-minus cells was not due to the imbalance between sigma factors caused by the lack of the protease. ftsH mutants could not grow in media with glutamine as the only nitrogen source and failed also to induce the sigma 54 promoters PnifH by NifA and PpspA by PspF. These lesions were fully complemented by a ftsH+ plasmid. Therefore, part of the pleiotropic phenotype of FtsH-less cells corresponded to the lack of sigma 54 activity. Overproduction of sigma 54, however, restored both transcriptional activity of Pu and growth in glutamine of a ftsH strain. These observations suggested that the activity of sigma 54 is checked in vivo by an interplay of factors that ultimately determine the performance of cognate promoters under given physiological conditions.