Two strains of Gram-negative, aerobic, non-pigmented, non-motile, rod-shaped bacteria were isolated from beech wood blocks during decay by the white-rot fungus Hypholoma fasciculare (Folman et al., 2008) and were designated strains BW863T and BW872. They are capable of methylotrophic growth and assimilate carbon via the ribulose-bisphosphate pathway. In addition to methanol, the novel isolates utilized ethanol, pyruvate and malate. Strains BW863T and BW872 are obligately acidophilic, mesophilic organisms capable of growth at pH values between 3.1 and 6.5 (with an optimum at pH 4.5-5.0) and at temperatures between 4 and 30 °C. Phospholipid fatty acid profiles of these bacteria contain unusually high amounts (about 90%) of 18:17c fatty acid, thereby resembling the profiles of Methylobacterium strains. The predominant quinone is Q-10. The DNA G+C content of novel isolates is 61.8-62.8 mol %. On the basis of 16S rRNA gene sequence identity, strains BW863T and BW872 are most closely related to the acidophilic methanotroph Methylocapsa acidiphila B2 (96.5-97 %). Comparative sequence analysis of mxaF, the gene encoding the large subunit of methanol dehydrogenase, placed the MxaF sequences of two novel strains in a cluster that is distinct from all previously described MxaF sequences of cultivated methylotrophs. The identity values between the MxaF sequences of the acidophilic isolates and the MxaF sequences from known alpha-, beta- and gammaproteobacterial methylotrophs comprised 69-75%, 61-63% and 64-67%, respectively. The data therefore suggest that strains BW863T and BW872 represent a novel genus and species of methylotrophic bacteria; the name Methylovirgula ligni gen. nov., sp. nov. is proposed, with strain BW863T (=DSM 19998T = NCIMB 14408T) as the type strain.