Among the fast-growing mycobacteria, members of the Mycobacterium fortuitum complex are the most-commonly cited opportunistic human pathogens, notably in post-surgical infections. Previous studies showed that this complex was composed of four well-identified species and a group of isolates that did not correspond to recognized species, which has been referred to as the third biovariant complex. The occurrence and chemical structure of the glycolipid antigens of six strains that belong to this latter group were examined in the present study. Based on the TLC profiles, resistance to alkali and seroreactivities of their glycolipids, the examined strains were classified into three groups: one group was devoid of species-specific glycolipid and the two other groups contained alkali-stable or alkali-labile glycoconjugates. The structures of the major glycolipid antigens of the latter two groups were elucidated by fastatom-bombardment MS, one-dimensional and two-dimensional NMR spectroscopy and conventional chemical analyses. The alkali-stable glycolipids were structurally identical to the C-mycoside-type glycopeptidolipids characterized in the taxonomically related species Mycobacterium peregrinum. The major alkali-labile glycolipid was identified as ~-Glcp-(1-+6)-a-Glcp2Acyl-(l-l)-a-Glcp3,4,6Acyl,. The acyl substituents consisted of one acetyl group and three fatty acyl residues composed mainly of tetradecanoyl residues, but significant amounts of 2-methylhexadecanoyl and 2-methyloctadecanoyl substituents were also present. The heterogeneity of the glycolipid content of members of the third biovariant complex of M. fortuitum demonstrated in the present study confirms the heterogeneity of the complex. In addition, the occurrence of a species-specific glycolipid in some strains supports the hypothesis that some strains of this complex of M. fortuitum may belong to a new mycobacterial species.Keywords: antigen ; glycopeptidolipid ; lipooligosaccharide ; mycobacteria.Interest in mycobacteria other than Mycobacterium tuberculosis has been renewed by the increased frequency of clinical infections caused by these organisms (Woods and Washington, 1987). Unlike M. tuberculosis, whose host is man, most other mycobacteria are environmental microorganisms widely distributed in nature. Among the fast-growing mycobacteria, the mostcommonly cited opportunistic human pathogens, notably in post-surgical infections, are members of the Mycobacterium fortuitum complex (Good, 198.5). Biovariants and subspecies of this complex have frequently been misidentified and can, with difficulty, be recognized as distinct species. Strains that belong to this complex are resistant to most antimycobacterial drugs, and their response to other antimicrobial agents depends on the species and biovariant or subspecies (Good, 1985). The the third biovariant (LCvy-Frtbault et al., 1983). The glycolipid content of M. fortuitum differs from that of M. peregrinum