Respiration of N oxides (denitrification) by bacteria is expressed facultatively in response to environmental stimuli. We have studied the transcriptional organization of the nos gene cluster of Pseudomonas stutzeri. This cluster carries the information for a functional nitrous oxide reductase (NosZ) which catalyzes the last step of the denitrification process. The nos genes are transcribed in three units, nosR, nosZ, and nosDFY. Transcription of nosZ is initiated from six different promoters which extend over a region of about 200 bp. The activity of two promoters varies subject to different growth conditions. Promoter P3 is active preferentially under denitrifying conditions and presumably under the control of a homolog of the transcriptional regulator FNR. Promoter P2 is the most active start site under aerobiosis and likely to initiate the low constitutive expression of nosZ. Transcription of nosR, encoding a regulator for nosZ expression, and transcription of the nosDFY operon, required for the copper chromophore assembly of NosZ, are both initiated from a single promoter. Transcription of nosR and the nosDFY operon was shown by phoA and lacZ fusions to be activated under a lowered oxygen tension and the simultaneous presence of an N oxide. The enzymatic activities associated with the hybrid proteins suggest for NosR and NosF a location in the cytoplasmic membrane and the cytoplasm, respectively.