A number of mycobacterial strains with similar growth characteristics, metabolic properties, and lipid compositions, which were previously placed in the Helsinki group (E. Brander, E. Jantzen, R. Huttunen, A. Juntunen, and M.-L. Katila, J. Clin. Microbiol. 30:1972-1975, 1992), were characterized by performing 16S rRNA gene sequencing. Of the 14 strains studied, 9 had a unique, previously undescribed sequence in the variable region of 16S rRNA. These nine strains, all of which were isolated from respiratory tract specimens, were nonpigmented and grew at 25 degrees C to 45 degrees C, reaching full colony size after 2 to 3 weeks. They produced arylsulfatase, nicotinamidase, and pyrazinamidase and were negative for Tween 80 hydrolysis, catalase, urease, and nitrate reductase activities, and niacin. Their glycolipid patterns were identical. A mycolic acid analysis performed by using thin-layer chromatography showed that these organisms contained alpha-mycolates, ketomycolates, and carboxy mycolates. Gas-liquid chromatography revealed that 2-eicosanol was the major alcohol and hexacosanoic acid was the major mycolic acid cleavage product. On the basis of their growth, biochemical, and lipid characteristics and their unique 16S rRNA sequence, we propose that these organisms should be assigned to a new species, Mycobacterium branderi. Comparative 16S rRNA sequencing revealed that this new species is closely related to Mycobacterium celatum, Mycobacterium cookii, and Mycobacterium xenopi. Strains 52157T (T = type strain) and 43548 have been deposited in the American Type Culture Collection as strains ATCC 51789 and ATCC 51788, respectively.