A novel moderately thermophilic methanotroph, strain MYHT(T), was isolated from a hot spring in Japan. The isolate grew on methane or methanol at 37-67 degrees C, and optimally at 57-59 degrees C. It was found to be a Gram-negative aerobe, with colourless colonies of non-motile coccoid cells, possessing type I intracytoplasmic membranes and regularly arranged surface layers of linear (p2) symmetry. Strain MYHT(T) expressed only the particulate methane monooxygenase and employed the ribulose monophosphate pathway for formaldehyde assimilation. It is a neutrophilic and halotolerant organism capable of growth at pH 6.5-7.5 (optimum pH 6.8) and in up to 3% NaCl (optimum 0.5-1% NaCl). Phylogenetic analysis based on 16S rRNA gene sequence analysis indicated that strain MYHT(T) is most closely related to the thermophilic undescribed methanotroph 'Methylothermus' HB (91% identity) and the novel halophilic methanotroph Methylohalobius crimeensis 10Ki(T) (90% identity). Comparative sequence analysis of particulate methane monooxygenase (pmoA) genes also confirmed the clustering of strain MYHT(T) with 'Methylothermus' HB and Methylohalobius crimeensis 10Ki(T) (98 and 92% derived amino acid sequence identity, respectively). The DNA G+C content was 62.5 mol%. The major cellular fatty acids were C(16:0) (37.2%) and C(18:1)omega9c (35.2%) and the major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The major ubiquinone was Q-8. On the basis of comparative phenotypic and genotypic characteristics, a new genus and species, Methylothermus thermalis gen. nov., sp. nov., is proposed, with MYHT(T) as the type strain (=VKM B-2345(T)=IPOD FERM P-19714(T)).