Fatty and mycolic acids and the pattern of glycolipids were studied in a collection of 34 strains of 'Mycobacterium habana' and in two strains of Mycobacterium simiae. Major glycolipids of these micro-organisms were assigned to the glycopeptidolipid (GPL) structural type, but both mycobacteria differed in the patterns obtained by TLC. The strains of 'M. habana' were separated into four groups (A-D), taking into account the presence or absence of several polar GPLs: group A contained GPL-I, GPL-ll and GPGIII; group B contained GPGI, GPGII', GPGll and GPGIII; group C contained GPGII', GPL-II and GPGIII; group D did not contain any of these compounds. Fatty acids of both bacteria were similar, and ranged from 14 to 26 carbon atoms, hexadecanoic, octadecenoic and tuberculostearic acids being predominant. Mycolic acids were also similar by TLC and HPLC, and consisted of a=, a'-and ketomycolates.Partial structural analysis by M S carried out in strains 'M. habana' TMC 5135 and M. simiae ATCC 25275l revealed that a-and ketomycolates ranged, in general, from 79 to 87 carbon atoms, and a'-mycolates from 58 to 67 carbon atoms. The a-and ketomycolates belonged to several structural series, and minor variations were found between the two strains examined. The data obtained justified the synonymy between 'M. habana' and M. simiae but indicated, in turn, that the former can be distinguished on the basis of GPL analysis. Most strains of 'M. habana' can be defined by the presence of GPGll and GPGIII, a finding that could be useful in the quality control of potential vaccine strains.