By transposon Tn5 mutagenesis, 19 strains of Pseudomonas stutzeri were acquired that had defects in nitrous oxide respiration (Nos^- phenotype). A physical map of the mutants showed nearly random Tn5 insertions into genomic DNA within a single region ca. 8 kilobases long. Mutants were characterized immunochemically, enzymatically, and chemically. Several functions related to the synthesis and regulation of nitrous oxide reductase were associated with this DNA region, indicating that in P. stutzeri part of the genetic information necessary to respire nitrous oxide is clustered.