PspF bound to the psp enhancer activates E sigma54 holoenzyme-dependent transcription of the Escherichia coli phage-shock protein (psp) operon and autogenously represses its own sigma70-dependent transcription, thereby keeping its concentration at a low level. It has been demonstrated previously that integration host factor (IHF) bound to a DNA site located between the psp core promoter and the PspF binding sites stimulates psp expression. We show here that wild-type IHF strongly retards DNA containing the psp promoter region. In vitro, PspF binding to the psp enhancer facilitates IHF binding, while IHF binding to the pspF-pspA-E promoter-regulatory region increases the efficacy of PspF binding to the upstream activating sequences (UASs). This is the first demonstration of co-operative binding of an activator and IHF in a sigma54-dependent system. In the absence of IHF, in vivo autoregulation of pspF transcription is lifted and, consequently, PspF production is increased, indicating that IHF enhances PspF binding to the psp enhancer in vivo.