After shifting an oxygen-respiring culture of Pseudomonas stutzeri to nitrate or nitrite respiration, we directly monitored the expression of the nirS gene by mRNA analysis. nirS encodes the 62-kDa subunit of the homodimeric cytochrome cd1 nitrite reductase involved in denitrification. Information was sought about the requirements for gene activation, potential regulators of such activation, and signal transduction pathways triggered by the alternative respiratory substrates. We found that nirS, together with nirT and nirB (which encode tetra- and diheme cytochromes, respectively), is part of a 3.4-kb operon. In addition, we found a 2-kb monocistronic transcript. The half-life of each of these messages was approximately 13 min in denitrifying cells with a doubling time of around 2.5 h. When the culture was subjected to a low oxygen tension, we observed a transient expression of nirS lasting for about 30 min. The continued transcription of the nirS operon required the presence of nitrate or nitrite. This anaerobically manifested N-oxide response was maintained in nitrate sensor (NarX) and response regulator (NarL) knockout strains. Similar mRNA stability and transition kinetics were observed for the norCB operon, encoding the NO reductase complex, and the nosZ gene, encoding nitrous oxide reductase. Our results suggest that a nitrate- and nitrite-responsive regulatory circuit independent of NarXL is necessary for the activation of denitrification genes.