Sequences of copper-containing nitrite reductase (nirK) genes obtained from completed nitrifier genome sequences were used to design polymerase chain reaction (PCR) primers to amplify partial nirK sequences from one Nitrosomonas and four Nitrosospira isolates. Deduced NirK protein sequences were highly similar to other copper-containing nitrite reductases including conserved motifs. Phylogenetic comparisons of NirK protein sequences placed orthologues from Nitrosomonas, Nitrosospira and Nitrobacter species into multiple distinct clades. Products related to nirK genes were not amplified from seven additional Nitrosomonas and Nitrosospira isolates by PCR with nirK-specific primers; however, DNA extracted from four of these isolates produced detectable signals in low-stringency Southern hybridizations probed with nirK gene fragments from ammonia-oxidizers with known nirK gene sequences. Analysis of promoter regions of nitrifier nirK genes revealed conserved binding motifs for the NsrR transcription factor in only one clade; other available nitrifier nirK gene promoters lacked characterized transcription factor binding motifs. Taken together, these results indicate that the sequences and regulation of nirK genes are diverse among nitrifiers. This study provides insight to the use of nirK genes for molecular diversity studies, establishes a framework to resolve the origins and diversification of nitrite reduction among the nitrifiers, and expands the database of nirK orthologues.