Slowly growing, scotochromogenic bacteria of a novel Mycobacterium species were isolated from lymph node samples in two children and pulmonary samples in two elderly patients from different regions in the Netherlands as well as from a surface water sample in Zambia. Its 16S rRNA gene, 16S-23S internal transcribed spacer (ITS), hsp65 and rpoB gene sequences are unique in comparison with other mycobacteria. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that these micro-organisms are most closely related to Mycobacterium scrofulaceum ATCC 19981(T) (8 differences; 0.6 % divergence). The hsp65 sequence shows 96 % similarity to that of Mycobacterium saskatchewanense MB54784 and the rpoB sequence shows 95 % similarity to that of Mycobacterium chimaera CIP 107892(T). The 16S-23S ITS sequence places these micro-organisms within the Mycobacterium avium complex, as a novel ITS sequevar. This is not supported by analysis of the 16S rRNA, hsp65 or rpoB gene sequences. Their scotochromogenicity, combined with mostly positive urease, positive semiquantitative catalase and negative tellurite reduction tests, set these isolates apart from related species. The mycolic acid patterns, obtained by HPLC, are similar to that of Mycobacterium scrofulaceum, though the peak heights and distribution present minor differences. We propose the name Mycobacterium mantenii sp. nov. for this novel species. The type strain, isolated from a lymph node biopsy sample, is strain 04-1474(T) (=NLA000401474(T) =CIP 109863(T) =DSM 45255(T)).