Methanotrophic bacteria were enumerated and isolated from the chemocline and surface sediments of marine-salinity Antarctic meromictic lakes located in the Vestfold Hills, Antarctica (68 degrees S 78 degrees E). Most probable number (MPN) analysis indicated that at the chemocline of Ace Lake the methanotroph population made up only a small proportion of the total microbial population and was sharply stratified, with higher populations detected in the surface sediments collected at the edge of Ace Lake and Burton Lake. Methanotrophs were not detected in Pendant Lake. Only a single phenotypic group of methanotrophs was successfully enriched, enumerated and isolated into pure culture from the lake samples. Strains of this group were non-motile, coccoidal in morphology, did not form resting cells, reproduced by constriction, and required seawater for growth. The strains were also psychrophilic, with optimal growth occurring at 10-13 degrees C and maximum growth temperatures of 16-21 degrees C. The ribulose monophosphate pathway but not the serine pathway for incorporation of C1 compounds was detectable in the strains. The guanine plus cytosine (G + C) content of the genomic DNA was 43-46 mol%. Whole-cell fatty acid analysis indicated that 16:1 omega 8c (37-41%), 16:1 omega 6c (17-19%), 16:1 omega 7c (15-19%) and 16:0 (14-15%) were the major fatty acids in the strains. 16s rDNA sequence analysis revealed that the strains form a distinct line of descent in the family Methylococcaceae (group I methanotrophs), with the closest relative being the Louisiana Slope methanotrophic mytilid endosymbiont (91.8-92.3% sequence similarity). On the basis of polyphasic taxonomic characteristics the Antarctic lake isolates represent a novel group I methanotrophic genus with the proposed name Methylosphaera hansonii (type strain ACAM 549).