The genetic organization of the nirD locus of Pseudomonas stutzeri ZoBell, necessary for a catalytically active cytochrome cd1 (EC 1.9.3.2), was determined. The locus comprises the unidirectionally transcribed open reading frames nirFDLGH, downstream of nirMC of the nir gene cluster, and immediately upstream of the norCB operon encoding nitric oxide (NO) reductase (EC 1.7.99.7). Notable sequence relatedness was found between NirF and cytochrome cd1 (NirS), within NirDLGH, and between NirM and NirC, suggesting several gene duplication events in this region. The derived NirF protein (391 amino acids, Mr 43,137) has 23.8% identity (51.1% overall similarity) with NirS, but lacks the N-terminal heme-c-binding domain of NirS. Insertional mutagenesis of the five open reading frames resulted in the loss of respiratory nitrite reductase activity in vivo and in vitro. Mutant strains, when induced with nitrate for denitrification, synthesized a periplasmic cytochrome cd1 lacking heme d1. The defect was caused by the inability of the cell to synthesize heme d1. The nirD locus is proposed to encode a multimeric and multifunctional enzyme complex involved in the synthesis of heme d1. Mutations in nirFDLGH lowered substantially the expression level of norCB. Nir- mutants, unable to generate NO in vivo, provide indirect evidence for an NO sensor and an inducer role of NO for its cognate reductase.