The NarL and NarP proteins are homologous response regulators that function to regulate anaerobic respiratory gene expression in response to nitrate and nitrite in Escherichia coli. Expression of the aeg-46.5 operon (anaerobically expressed gene at 46.5 minutes on the genetic map) is induced during anaerobic growth by the global transcriptional regulatory protein Fnr. aeg-46.5 operon expression is further induced by the NarP protein in response to nitrate or nitrite and this induction is antagonized by NarL. We used in vivo and in vitro techniques to investigate how these three transcriptional regulatory proteins control the activity of a single promoter. Deletion and mutational analysis of the aeg-46.5 operon control region identified two distinct cis-acting elements. A sequence with similarity to the Fnr-binding site consensus, centered at position -64.5, was essential for Fnr-dependent anaerobic induction of aeg-46.5 operon expression. In all other naturally occurring Fnr-dependent promoters the primary Fnr-binding site is centered between -40 and -50. The second cis-acting element, a region of perfect symmetry centered at -44.5, shares sequence similarity with the NarL-binding site consensus. This region was required for nitrate and nitrite induction of aeg-46.5 operon expression. We purified the NarP and NarL proteins as maltose-binding protein (MBP) fusion proteins and investigated their interaction with the aeg-46.5 operon control region. Incubation with the phospho-donor, acetyl phosphate, allowed both MBP-NarP and MBP-NarL to protect the -44.5 region of the aeg-46.5 operon control region from DNase I cleavage. Single and double nucleotide substitutions in the -44.5 region reduced or abolished nitrate and nitrite induction of aeg-46.5 operon expression in vivo and prevented the binding of MBP-NarP and MBP-NarL to the control region in vitro. Presumably, the NarP and NarL proteins compete for the -44.5 binding site to regulate aeg-46.5 operon expression in response to nitrate and nitrite. Apparently, only the NarP protein is competent to activate transcription of the aeg-46.5 operon when bound to the -44.5 region.