The nirC and nirF genes were identified downstream from nirSM, the structural genes encoding nitrite reductase (NIR) and cytochrome c-551 from Pseudomonas aeruginosa (Pa). The nirC gene encodes a probable c-type cytochrome with a signal sequence for membrane translocation. The nirF gene codes for a protein of 392 amino acids. A nirF mutant of Pa, constructed by marker exchange mutagenesis, synthesized an inactive NIR protein whose activity was restored by adding purified heme d1. The mutant strain produced an active NIR, when it was transformed by a broad-host-range plasmid carrying nirF. These results showed that the product of nirF was essential for the biosynthesis of heme d1 in Pa.