Genes encoding particulate methane monooxygenase (pMMO) and ammonia monooxygenase (AMO) show high sequence identity. Degenerate primers were designed based on shared amino acid sequences of the 27-kDa polypeptides, which are believed to contain active sites. PCR amplified, cloned, and sequenced DNA fragments from various methanotrophic and nitrifying bacteria. Analysis of the predicted amino acid sequences revealed strong conservation of primary and secondary structures. Nitrosococcus oceanus AmoA showed higher identity to γ-Proteobacteria PmoA than to β-Proteobacteria AmoA. These results suggest that pMMO and AMO are evolutionarily related despite their different physiological roles. The study included representatives of all phylogenetic groups of methanotrophs (α- and γ-Proteobacteria) and ammonia-oxidizing nitrifiers (β- and γ-Proteobacteria). The pmoA and amoA genes were amplified using specific primers, and sequences were analyzed. The high level of identity between pMMO and AMO gene products suggests a common ancestry. The conservation of amino acid sequences correlated with phylogenetic relatedness rather than functional differences. The study confirmed the presence of pmoA and amoA genes in all methanotrophs and nitrifiers examined, extending the database of available sequences. The strong conservation of primary and secondary structures of PmoA and AmoA from diverse organisms suggests these genes share a common ancestry. The correlation of sequence identity clusters with phylogenetic affiliations further supports the evolutionary relationship between pMMO and AMO. Despite their different roles in methanotrophs and ammonia-oxidizing nitrifiers, these enzymes may be considered evolutionary homologues.Genes encoding particulate methane monooxygenase (pMMO) and ammonia monooxygenase (AMO) show high sequence identity. Degenerate primers were designed based on shared amino acid sequences of the 27-kDa polypeptides, which are believed to contain active sites. PCR amplified, cloned, and sequenced DNA fragments from various methanotrophic and nitrifying bacteria. Analysis of the predicted amino acid sequences revealed strong conservation of primary and secondary structures. Nitrosococcus oceanus AmoA showed higher identity to γ-Proteobacteria PmoA than to β-Proteobacteria AmoA. These results suggest that pMMO and AMO are evolutionarily related despite their different physiological roles. The study included representatives of all phylogenetic groups of methanotrophs (α- and γ-Proteobacteria) and ammonia-oxidizing nitrifiers (β- and γ-Proteobacteria). The pmoA and amoA genes were amplified using specific primers, and sequences were analyzed. The high level of identity between pMMO and AMO gene products suggests a common ancestry. The conservation of amino acid sequences correlated with phylogenetic relatedness rather than functional differences. The study confirmed the presence of pmoA and amoA genes in all methanotrophs and nitrifiers examined, extending the database of available sequences. The strong conservation of primary and secondary structures of PmoA and AmoA from diverse organisms suggests these genes share a common ancestry. The correlation of sequence identity clusters with phylogenetic affiliations further supports the evolutionary relationship between pMMO and AMO. Despite their different roles in methanotrophs and ammonia-oxidizing nitrifiers, these enzymes may be considered evolutionary homologues.