Production of nitric oxide (NO) and nitrous oxide (N2O) by ammonia (NH3)-oxidizing bacteria in natural and man-made habitats is thought to contribute to the undesirable emission of NO and N2O into the earth's atmosphere. The NH3-oxidizing bacterium Nitrosomonas europaea expresses nitrite reductase (NirK), an enzyme that has so far been studied predominantly in heterotrophic denitrifying bacteria where it is involved in the production of these nitrogenous gases. The finding of nirK homologues in other NH3-oxidizing bacteria suggests that NirK is widespread among this group; however, its role in these nitrifying bacteria remains unresolved. We identified a gene, nsrR, which encodes a novel nitrite (NO2–)-sensitive transcription repressor that plays a pivotal role in the regulation of NirK expression in N. europaea. NsrR is a member of the Rrf2 family of putative transcription regulators. NirK was expressed aerobically in response to increasing concentrations of NO2– and decreasing pH. Disruption of nsrR resulted in the constitutive expression of NirK. NsrR repressed transcription from the nirK gene cluster promoter (Pnir), the activity of which correlated with NirK expression. Reconstruction of the NsrR-Pnir system in Escherichia coli revealed that repression by NsrR was reversed by NO2– in a pH-dependent manner. The findings are consistent with the hypothesis that N. europaea expresses NirK as a defence against the toxic NO2– that is produced during nitrification.