In Escherichia coli K-12 the expression of many genes is controlled by the oxygen-responsive transcription factor FNR and the nitrate- and nitrite-responsive two-component systems NarXL and NarPQ. Here, the ydhY gene is shown to be the first gene, of a six-gene operon (ydhYVWXUT) that encodes proteins predicted to be components of an oxidoreductase. Mapping the ydhY-T transcript start and site-directed mutagenesis confirmed that the ydhY-T genes are transcribed from an FNR-dependent class II promoter and showed that the FNR site is centred at -42.5. In the presence of nitrate or nitrite, NarXL and NarPQ repressed ydhY-T expression. Analysis of the DNA sequence of the ydhY promoter region (PydhY) revealed the presence of four heptameric sequences similar to NarL/P binding sites centred at -42, -16, +6 and +15. The latter heptamers are arranged as a 7-2-7 inverted repeat, which is required for recognition by NarP. Accordingly, NarP protected the 7-2-7 region in DNase I footprints, and mutation of either heptamer +6 or heptamer +15 impaired nitrite-mediated regression, whereas mutation of heptamer -42 and heptamer -16 did not affect the response to nitrite. The NarL protein also protected the 7-2-7 region, but in contrast to NarP, the NarL footprint extended further upstream to encompass the -16 heptamer. The extended NarL footprint was consistent with the presence of multiple NarL-PydhY complexes in gel retardation assays. Mutation of heptamer -42, which is located within the FNR binding site, or heptamer + 6 (but not heptamers -16 or +15) impaired nitrate-mediated repression. Thus, although the region of the ydhY-T promoter containing the -16 and +15 heptamers was recognized by NarL in vitro, mutation of these heptamers did not affect NarL-mediated repression in vivo.