Fatty acids, alcohols, and mycolic acid cleavage products were determined for 13 ATCC strains and 24 clinical isolates, which were initially identified by biochemical and growth characteristics as the Mycobacterium terrae complex. The clinical isolates were also analyzed by partial sequencing of the 16S rRNA gene, which divided them into five genetic entities, M. triviale (three strains), M. terrae (four strains), M. nonchromogenicum sensu stricto (seven strains), Mycobacterium sp. strain MCRO 6 (seven strains), and Mycobacterium sp. strain 31958 (one strain). After acidic methanolysis, secondary alcohols were a characteristic feature in all members of the M. terrae complex but M. triviale. In addition to the prominent secondary alcohols, 2-octadecanol and 2-eicosanol, two previously unidentified alcohols, 2-(8,15-dimethyl)docosenol and 2-(8,17-dimethyl)tetracosenol, were detected in M. nonchromogenicum, Mycobacterium sp. strain MCRO 6, and Mycobacterium sp. strain 31958. Only 2-(8,17-dimethyl)tetracosenol was detected in trace amounts in M. terrae. Genetic differences were associated with differences in phenotypic characteristics, including growth at 42 degrees C and pyrazinamidase production. Based on fatty acid and alcohol composition and biochemical and genetic characteristics, M. non-chromogenicum and Mycobacterium sp. strains MCRO 6 and 31958 were found to be a closely related group, named the M. nonchromogenicum complex. Detected genetic variations associated with phenotypic characteristics may indicate further species separation of this complex. In conclusion, the results of gas-liquid chromatography fatty acid analysis, combined with those of a Tween 80 test, enable identification of the species of the M. terrae complex and their separation from other nonpigmented slowly growing mycobacteria.