A novel methane-oxidizing bacterium, strain HTM55(T), was isolated from subsurface hot aquifer water from a Japanese gold mine. Strain HTM55(T) was a Gram-negative, aerobic, motile, coccoid bacterium with a single polar flagellum and the distinctive intracytoplasmic membrane arrangement of a type I methanotroph. Strain HTM55(T) was a moderately thermophilic, obligate methanotroph that grew on methane and methanol at 37-65 °C (optimum 55-60 °C). The isolate grew at pH 5.2-7.5 (optimum 5.8-6.3) and with 0-1 % NaCl (optimum 0-0.3 %). The ribulose monophosphate pathway was operative for carbon assimilation. The DNA G+C content was 54.4 mol% and the major fatty acids were C(16 : 0) (52.0 %) and C(18 : 1)ω7c (34.8 %). Phylogenetic analysis of the 16S rRNA gene sequence indicated that strain HTM55(T) was closely related to Methylothermus thermalis MYHT(T) (99.2 % 16S rRNA gene sequence similarity), which is within the class Gammaproteobacteria. However, DNA-DNA relatedness between strain HTM55(T) and Methylothermus thermalis MYHT(T) was ≤ 39 %. On the basis of distinct phylogenetic, chemotaxonomic and physiological characteristics, strain HTM55(T) represents a novel species of the genus Methylothermus, for which the name Methylothermus subterraneus sp. nov. is proposed. The type strain is HTM55(T) ( = JCM 13664(T) = DSM 19750(T)).