Five independent strains, isolated from clinical samples but probably not responsible for disease, revealed phenotypic and genotypic features that appeared to exclude their belonging to any of the recognized Mycobacterium species. The strains, which are non-pigmented rapid growers, presented a cell-wall lipid pattern resembling those of Mycobacterium brumae and Mycobacterium fallax. Sequencing of the 16S rRNA, hsp65 and rpoB genes and the 16S-23S rRNA gene internal transcribed spacer (ITS) revealed that the strains are clearly distinct from every other Mycobacterium species. While the 16S rRNA and rpoB genes were characterized by a single sequevar, two sequevars were detected in hsp65 and three in the ITS. The divergence shown in the latter region was striking, in which only two short regions (less than 150 bp in all) were comparable with other mycobacteria, apart from Mycobacterium monacense and Mycobacterium gilvum. The PCR restriction analysis pattern of the novel strains also differs from any reported to date. The name Mycobacterium insubricum sp. nov. is proposed for the novel species; the type strain is FI-06250(T) (=DSM 45132(T) =CIP 109609(T)).