The stimulation of Fnr-dependent transcription from the narG promoter by NarL-phosphate is known to require a cis-acting sequence, the NarL box, located approximately 195 bp upstream from the transcription start site, and the interaction of integration host factor (IHF) with a binding site in the intervening region (positions -110 to -140) between the NarL box and the transcription start site. By gel retardation and DNase I protection studies, we have demonstrated that NarL-phosphate, produced by the reaction of purified NarL with acetyl phosphate, specifically binds to a fragment derived from the upstream region of the narG promoter. The fragment was protected by NarL-phosphate binding to two distinct regions. One was an extended sequence of approximately 40 bp surrounding the NarL box at -195; the second was located downstream from the IHF-binding region and included a sequence extending from positions -80 to -120. Alteration by site-directed mutagenesis of a putative inverted NarL box sequence identified within the downstream protected region in a plasmid containing a narG-lacZ fusion eliminated the NarL-phosphate-mediated stimulation of transcription. NarL-phosphate bound to the two regions independently from IHF binding and it bound to each site independently when the two sites were separated by cleavage of the promoter fragment. Stimulation of transcription from the narG promoter by NarL-phosphate appears to result from the formation of a folded protein-DNA structure created by the binding of NarL-phosphate to multiple sites on either side of an IHF-induced bend in the upstream region of the promoter.