Hyphomicrobium chloromethanicum CM2^T, an aerobic methylotrophic member of the  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 C₁ transfer pathway for carbon assimilation and CO₂ 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.