~~~~ ~ Two-dimensional thin-layer chromatography of whole-organism acid methanolysates of Mycqbacterium chelonei gives a characteristic pattern of two non-polar mycolic acid methyl esters which allows the organism to be distinguished from all other mycobacteria including Mycobacterium fortuitum. The mycolic acids from Mycobacterium chelonei were composed of approximately equal amounts of a diunsaturated a-mycolate and a lower molecular weight a'-mycolate, though minor amounts of a different a-mycolate were also detected. These mycolic acids are the first examples of natural mixtures from mycobacteria lacking major amounts of acids having oxygen functions in addition to the 3-hydroxy acid unit.