Laboratory strains of Mycobacterium phlei, M. smegmatis, M. fortuitum, M. gordonae, M. kansasii, M. bovis, M. tuberculosis and M. intracellulare were adapted to grow in an anaerobic environment. Concomitant with the transition to anaerobic growth was loss of acid-fastness, loss or modification of colonial pigmentation, and loss of ability of grow on a malachite green-containing medium. The mycobacteria grown anaerobically produced acid from a greater range of carbohydrates than aerobically grown cultures, lost iron-uptake activity, and showed a reduction of urease catalase and nitratase activity. Black adaption of mycobacteria from an anaerobic to an aerobic environment resulted in the acquisition of acid-fastness, pigmentation, and other characteristics used in the taxonomy of mycobacteria. These results suggest that mycobacterial cultures, if grown in an anaerobic environment, may be erroneously identified in clinical laboratories.