The ability of methylotrophic alpha-proteobacteria to grow with dichloromethane (DCM) as source of carbon and energy has long been thought to depend solely on a single cytoplasmic enzyme, DCM dehalogenase, which converts DCM to formaldehyde, a central intermediate of methylotrophic growth. The gene dcmA encoding DCM dehalogenase of Methylobacterium dichloromethanicum DM4 was expressed from a plasmid in closely related Methylobacterium strains lacking this enzyme. The ability to grow with DCM could be conferred upon Methylobacterium chloromethanicum CM4, a chloromethane degrader, but not upon Methylobacterium extorquens AM1. In addition, growth of strain AM1 with methanol was impaired in the presence of DCM. The possibility that single-carbon (C1) utilization pathways in dehalogenating Methylobacterium strains differed from those discovered in strain AM1 was addressed. Homologues of tetrahydrofolate-linked and tetrahydromethanopterin-linked C1 utilization genes of strain AM1 were detected in both strain DM4 and strain CM4, and cloning and sequencing of several of these genes from strain DM4 revealed very high sequence identity (96.5-99.7%) to the corresponding genes of strain AM1. The expression of transcriptional xylE fusions of selected genes of the tetrahydrofolate- and tetrahydromethanopterin-linked pathways from strain DM4 was investigated. The data obtained suggest that the expression levels of some C1 utilization genes in M. dichloromethanicum DM4 grown with DCM may differ from those observed during growth with methanol.