Experiments were conducted to determine if a novel pmoA-like gene (pmoA2) recently discovered in the methane-oxidizing bacterium Methylocystis strain SC2 (P. F. Dunfield, M. Tchawa Yimga, S. D. Dedysh, U. Berger, W. Liesack, and J. Heyer, FEMS Microbiol. Ecol. 41:17-26, 2002) is present in other methane-oxidizing bacteria (MOB), and if it is expressed. A newly developed primer combination (pmoA206f-pmoA703b) allowed a differential detection of pmoA1 and pmoA2. By using this primer combination, we identified pmoA2 in a wide range of type II MOB of the Methylosinus-Methylocystis group. However, screening by PCR and by Southern hybridization using a newly developed pmoA2-specific oligonucleotide probe also showed that closely related type II MOB, exhibiting 16S rRNA gene sequence identities of higher than 97%, may or may not harbor pmoA2. No pmoA2 was detected in five type I MOB tested: Methylococcus capsulatus strain Bath, Methylocaldum strain E10A, Methylobacter luteus, Methylomicrobium album, and Methylomonas strain D1a. In comparative sequence analyses, all pmoA2-like sequences formed a coherent cluster clearly distinct from pmoA1 sequences of type I and type II MOB, and from amoA sequences of the Nitrosomonas-Nitrosospira group. Phylogenetic analysis using the paml model suggested that pmoA2 is subject to strong purifying selection and therefore has an important cellular function. We probed total RNA extracts of Methylocystis strain SC2 for gene expression of pmoA. A strong signal was observed for pmoA1 in Northern hybridization, while the results obtained for pmoA2 were ambiguous. However, reverse transcription-PCR confirmed that pmoA2 was expressed, albeit at lower level than pmoA1. This provided experimental evidence that the gene product of pmoA2 may be a functionally active enzyme.