Seven strains of the genus Sphaerotilus were obtained from natural thermal sulfide (strains D-501T, D-502, D-504, D-505 and D-507) and low-temperature ferrous (strain HST) springs and from an activated sludge system (strain D-380). These Sphaerotilus isolates and strains of Sphaerotilus natans obtained from the DSMZ (S. natans DSM 6575T, DSM 565 and DSM 566) were studied using a polyphasic taxonomic approach. All strains had Q-8 as the major quinone and C16 : 1ω7, C16 : 0 and C18 : 1ω7 as the major fatty acids. The DNA–DNA hybridization results and 16S rRNA, hsp60 and gyrB gene sequencing experiments showed that isolates D-501T, D-502, D-504, D-505, D-507 and D-380 were closely related to the type strain of S. natans DSM 6575T. However, strains D-501T, D-502, D-504, D-505 and D-507 significantly differed from the heterotrophic strain S. natans DSM 6575T by their capability for lithotrophic growth with reduced sulfur compounds as an electron donor for energy conservation and some other phenotypic features. For this reason, strains D-501T, D-502, D-504, D-505 and D-507 merit a separate taxonomic classification at the subspecies level. The name Sphaerotilus natans subsp. sulfidivorans subsp. nov. (type strain D-501T = DSM 22545T = VKM B-2573T) is proposed. The subspecies Sphaerotilus natans subsp. natans subsp. nov. is automatically created as a result of this proposal. Strain D-380 was phenotypically closely related to S. natans DSM 6575T. Strains D-380 and S. natans DSM 6575T were assigned to the subspecies Sphaerotilus natans subsp. natans subsp. nov. (type strain DSM 6575T = ATCC 13338T). The 16S rRNA, hsp60 and gyrB gene sequences obtained for strains HST and DSM 565 showed very low sequence similarity values of 97.3 %, 89.7 % and 88.4 %, respectively, with S. natans DSM 6575T. Strain HST shared 99 % DNA–DNA relatedness with strain