The primary structure of an nir gene cluster necessary for production of active dissimilatory nitrite reductase was determined from Pseudomonas aeruginosa. Seven open reading frames, designated nirDLGHJEN, were identified downstream of the previously reported nirSMCF genes. From nirS through nirN, the stop codon of one gene and the start codon of the next gene were closely linked, suggesting that nirSMCFDLGHJEN are expressed from a promoter which regulates the transcription of nirSM. The amino acid sequences deduced from the nirDLGH genes were homologous to each other. A gene, designated nirJ, which encodes a protein of 387 amino acids, showed partial identity with each of the nirDLGH genes. The nirE gene encodes a protein of 279 amino acids homologous to S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase from other bacterial strains. In addition, NirE shows 21.0% identity with NirF in the N-terminal 100-amino-acid residues. A gene, designated nirN, encodes a protein of 493 amino acids with a conserved binding motif for heme c (CXXCH) and a typical N-terminal signal sequence for membrane translocation. The derived NirN protein shows 23.9% identity with nitrite reductase (NirS). Insertional mutation and complementation analyses showed that all of the nirFDLGHJE genes were necessary for the biosynthesis of heme d1.