The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I， nothing is known about the insertion of the haem d1 into NirS. Here， we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS-NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further， we show by affinity co-purification that NirS also directly interacts with NirF. Additionally， NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally， the analysis by UV-visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results， we propose a potential model for the maturation of NirS in which the three proteins NirS， NirN and NirF form a transient， membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS.