Spiroplasma sp. strain GNAT3597T was isolated from the biting midge genus Atrichopogon (Diptera: Ceratopogonidae). It was serologically distinct from other Spiroplasma species, groups or subgroups. Dark-field microscopy of the cells revealed the classical helical shape and subsequent transmission electron microscopy revealed cells surrounded by only a cell membrane (i.e. lacking a cell wall). Growth of strain GNAT3597T occurred in M1D medium at 30 °C. Strain GNAT3597T catabolized both glucose and arginine, but did not hydrolyse urea. The DNA G+C content of strain GNAT3597T was 29±1 mol%. Only one strain, SMCAT (Spiroplasma mirum), is serologically related to strain GNAT3597T, although the relationship is weak (positive reaction to only a 1 : 80 dilution). It is therefore proposed that strain GNAT3597T (=ATCC BAA-520T=NBRC 100390T) represents a novel species, Spiroplasma atrichopogonis sp. nov. (class Mollicutes: order Entomoplasmatales: family Spiroplasmataceae).