Mycobacterium fortuitum, biovar, fortuitum, the cause of serious skin and soft-tissue infections, can be differentiated from M. fortuitum, biovar. peregrinum, and other rapidly growing opportunistic mycobacteria by the presence of a unique antigenic glycolipid. The glycolipid is among the simplest of the acyl-trehalose-containing lipooligosaccharide class. The application of 1H and 13C NMR, methylation analysis, FAB/MS, and other procedures demonstrated the structure, beta-D-Glcp-(1----6)-2-O-acyl-alpha-D-Glcp-(1 in equilibrium with 1)-3,4,6-tri-O-acyl-alpha-D-Glcp. Thus, practically all environmental mycobacteria, many of them opportunistic pathogens, can be differentiated serologically and chemically on the basis of unique sugar arrangements within a few classes of glycolipids. The simplicity of the structure in M. fortuitum fortuitum combined with the distinct roughness of the parent strain raises the intriguing possibility that it is a spontaneous rough variant of the other mycobacteria with more elaborate glycolipids.