ABSTRACT Hyphomicrobium chloromethanicum CM2 ^T , an aerobic methylotrophic member of the α subclass of the class proteobacteria , can grow with chloromethane as the sole carbon and energy source. H. chloromethanicum possesses an inducible enzyme system for utilization of chloromethane, in which two polypeptides (67-kDa CmuA and 35-kDa CmuB) are expressed. Previously, four genes, cmuA , cmuB , cmuC , and purU , were shown to be essential for growth of Methylobacterium chloromethanicum on chloromethane. The cmuA and cmuB genes were used as probes to identify homologs in H. chloromethanicum . A cmu gene cluster (9.5 kb) in H. chloromethanicum contained 10 open reading frames: folD (partial), pduX , orf153 , orf207 , orf225 , cmuB , cmuC , cmuA , fmdB , and paaE (partial). CmuA from H. chloromethanicum (67 kDa) showed high identity to CmuA from M. chloromethanicum and contains an N-terminal methyltransferase domain and a C-terminal corrinoid-binding domain. CmuB from H. chloromethanicum is related to a family of methyl transfer proteins and to the CmuB methyltransferase from M. chloromethanicum . CmuC from H. chloromethanicum shows identity to CmuC from M. chloromethanicum and is a putative methyltransferase. folD codes for a methylene-tetrahydrofolate cyclohydrolase, which may be involved in the C ₁ transfer pathway for carbon assimilation and CO ₂ production, and paaE codes for a putative redox active protein. Molecular analyses and some preliminary biochemical data indicated that the chloromethane utilization pathway in H. chloromethanicum is similar to the corrinoid-dependent methyl transfer system in M. chloromethanicum . PCR primers were developed for successful amplification of cmuA genes from newly isolated chloromethane utilizers and enrichment cultures.