The methanol dehydrogenase gene mxaF, encoding the large subunit of the enzyme, was amplified from the DNA of a number of representative methanotrophs, methyletrophs, and environmental samples by PCR using primers designed from regions of conserved amino acid sequence identified by comparison of three known sequences of the large subunit of methanol dehydrogenase. The resulting 550-bp PCR products were cloned and sequenced. Analysis of the predicted amino acid sequences corresponding to these mxaF genes revealed strong sequence conservation. Of the 172 amino acid residues, 47% were conserved among all 22 sequences obtained in this study. Phylogenetic analysis of these MxaF sequences showed that those from type I and type II methanotrophs form two distinct clusters and are separate from MxaF sequences of other gram-negative methylotrophs. MxaF sequences retrieved by PCR from DNA isolated from a blanket bog peat core sample formed a distinct phylogenetic cluster within the MxaF sequences of type II methanotrophs and may originate from a novel group of acidophilic methanotrophs which have yet to be cultured from this environment.