Microbial growth coupled to O-demethylation of phenyl methyl ethers, which are lignin decomposition products, was described for acetogenic bacteria and recently also for two species belonging to the nonacetogenic genus Desulfitobacterium. To elucidate the potential role of desulfitobacteria in the O-demethylation of phenyl methyl ethers in the environment, we cultivated Desulfitobacterium chlororespirans, D. dehalogenans, D. metallireducens, and different strains of D. hafniense with phenyl methyl ethers as sole electron donors. With the exception of D. metallireducens, all species and strains tested were able to demethylate at least three of the four phenyl methyl ethers applied with fumarate, nitrate, or thiosulfate as electron acceptor. Furthermore, a high number of operons putatively encoding demethylase systems were identified in the genomes of Desulfitobacterium spp., although discrimination between O-, S-, N- and, Cl-demethylases was not possible. These findings provide evidence for the importance of the methylotrophic metabolism for desulfitobacteria and point to their involvement in the O-demethylation of phenyl methyl ethers in the environment.