Representative strains of Mycobacterium fortuitum, 'Mycobacterium peregrinum' and Mycobacterium smegmatis were degraded by acid methanolysis and patterns of long-chain compounds were determined by two-dimensional thin-layer chromatography. The same general pattern of mycolic acid methyl esters was found in all 39 strains examined, the major components being so-called alpha-mycolates and characteristic pairs of polar mycolates. Analysis of alkaline methanolysates of selected strains confirmed that these polar mycolates were derived from epoxymycolic acids, as found previously.