Sixty-one strains of Mycobacterium tuberculosis complex and 47 strains of nontuberculous mycobacteria were analyzed for fatty acids and enzyme profiles. Cellular fatty acids were extracted from bacteria, methylated and analyzed by gas liquid chromatography operated either manually (Perkin-Elmer) or by the automatic Microbial Identification System. The major cellular fatty acids in all mycobacterial species were C16: 0 and C18: 1. Tuberculostearic acid was found in all species with the exception of Mycobacterium gordonae. The fatty acids with a carbon-length longer than 20 could be detected only by conventional gas chromatography. Strains of M. tuberculosis had a high ratio of C26: 0 to C24: 0, and a relatively low ratio of C14: 0 to C15: 0. For determination of branched-chain fatty acids, the MIS provided more definitive results. The data indicated that the fatty acid profiles could provide rapid species identification. The results of the enzyme profile analysis using API-ZYM strips showed 39 different patterns from 59 strains of M. tuberculosis, and 41 different patterns from 46 nontuberculous mycobacteria strains, suggesting that enzyme profiles can also be used for strain characterization within the same species.