Representative strains of some species of Mycobacterium were degraded by both acid and alkaline methanolysis. Two-dimensional thin-layer chromatography was used to determine the patterns of mycolic acids and other long-chain components in these methanolysates. Patterns composed of alpha-, methoxy- and ketomycolates were found in Mycobacterium asiaticum, Mycobacterium bovis, Mycobacterium gastri, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium marinum and Mycobacterium tuberculosis; a representative of Mycobacterium thermoresistibile also contained lower molecular weight alpha'-mycolates in addition to these three acids. In representatives of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium nonchromogenicum, "Mycobacterium novum", Mycobacterium paratuberculosis, Mycobacterium scrofulaceum, Mycobacterium terrae, Mycobacterium xenopi, and Mycobacterium sp. MNC 165 alpha- and ketomycolates were accompanied by omega-carboxymycolates and 2-eicosanol and homologous alcohols which are derived from wax-ester mycolates. Mycobacterium fortuitum and "Mycobacterium giae" contained alpha'- and epoxymycolates and both serovars of Mycobacterium simiae had a very characteristic pattern of alpha-, alpha'- and ketomycolic acids. Comparison with data for other mycobacteria showed the chemotaxonomic significance of these mycolic acid patterns.