A novel species is proposed for two strains of methanotrophic bacteria (H2T and Sakb1) isolated from an acidic (pH 4.3) Sphagnum peat bog lake (Teufelssee, Germany) and an acidic (pH 4.2) tropical forest soil (Thailand), respectively. Cells of strains H2T and Sakb1 were aerobic, Gram-negative, non-motile, straight or curved rods that were covered by large polysaccharide capsules and contained an intracytoplasmic membrane system typical of type II methanotrophs. They possessed both a particulate and a soluble methane monooxygenase and utilized the serine pathway for carbon assimilation. They were moderately acidophilic organisms capable of growth between pH 4.4 and 7.5 (optimum 5.8–6.2). The most unique characteristic of these strains was the phospholipid fatty acid profile. In addition to the signature fatty acid of type II methanotrophs (18 : 18c), the cells also contained large amounts of what was previously considered to be a signature fatty acid of type I methanotrophs, 16 : 18c. The DNA G+C contents of strains H2T and Sakb1 were 61.5 and 62.1 mol%, respectively. The 16S rRNA gene sequences possessed 96–98 % similarity to sequences of other type II methanotrophs in the genera Methylosinus and Methylocystis. 16S rRNA gene sequence and pmoA phylogeny demonstrated that the strains form a novel lineage within the genus Methylocystis. DNA–DNA hybridization values of strain H2T with Methylocystis parvus OBBPT and Methylocystis echinoides IMET 10491T were 18 and 25 %, respectively. Thus, it is proposed that these two strains represent a novel species, Methylocystis heyeri sp. nov. Strain H2T (=DSM 16984T=VKM B-2426T) is the type strain.