<jats:p>A novel methanotroph, strain LC 2<jats:sup>T</jats:sup>, was isolated from the littoral sediment of Lake Constance by enrichment in opposing gradients of methane and oxygen, followed by traditional isolation methods. Strain LC 2<jats:sup>T</jats:sup> grows on methane or methanol as its sole carbon and energy source. It is a Gram-negative, non-motile, pale-pink-coloured methanotroph showing typical intracytoplasmic membranes arranged in stacks. Cells are coccoid, elliptical or rod-shaped and occur often in pairs. Strain LC 2<jats:sup>T</jats:sup> grows at low oxygen concentrations and in counter-gradients of methane and oxygen. It can grow on medium free of bound nitrogen, possesses the <jats:italic>nifH</jats:italic> gene and fixes atmospheric nitrogen at low oxygen pressure. It grows at neutral pH and at temperatures between 10 and 30 °C. Phylogenetically, it is most closely related to the genus <jats:italic>Methylobacter</jats:italic>, with the type strains of <jats:italic>Methylobacter tundripaludum</jats:italic> and <jats:italic>Methylobacter psychrophilus</jats:italic> showing 94 and 93.4 % 16S rRNA gene sequence similarity, respectively. Furthermore, the <jats:italic>pmoA</jats:italic> gene sequence of strain LC 2<jats:sup>T</jats:sup> is most closely related to <jats:italic>pmoA</jats:italic> gene sequences of <jats:italic>Methylobacter</jats:italic> strains (92 % similar to <jats:italic>Methylobacter</jats:italic> sp. LW 12 by deduced amino acid sequence identity). The DNA G+C content is 49.9 mol% and the major cellular fatty acid is 16 : 1<jats:italic>ω</jats:italic>7<jats:italic>c</jats:italic> (60 %). Strain LC 2<jats:sup>T</jats:sup> (=JCM 14076<jats:sup>T</jats:sup>=DSM 18750<jats:sup>T</jats:sup>) is described as the type strain of a novel species within a new genus, <jats:italic>Methylosoma difficile</jats:italic> gen. nov., sp. nov.</jats:p>