A crude glycolipid fraction was isolated from Mycobacterium smegmatis ATCC 356 by ethanolic extraction and silica gel chromatography. After deacylation of the glycolipid fraction, a dipyruvylated pentasaccharide (acidic oligosaccharide A) and a monopyruvylated tetrasaccharide (acidic oligosaccharide B1) were purified by ion exchange and gel filtration chromatography. Methylation analysis, proton nuclear magnetic resonance, mass spectrometry, and periodate oxidation suggested that oligosaccharide A was 4,6-(1-carboxyethylidene)-3-O-methyl-beta-D Glcp-(1-3)-4,6-(1-carboxyethylidene)-beta-D-Glcp-(1-4)-beta-D-Glcp-(1-6)-alpha- D Glcp-(1-1)-alpha-D-Glcp and that oligosaccharide B1 was 4,6-(1-carboxyethylidene)-beta-D-Glcp-(1-4)-beta-D-Glcp-(1-6)-alpha-D-Glcp-(1-1 ) -alpha-D-Glcp. Both compounds contain a trehalose unit as a part of the oligosaccharide structure, and B1 appears to be a biosynthetic precursor of A because the two differ only in a pyruvylated 3-O-methylglucose unit. A third component, acidic oligosaccharide B2, differs from oligosaccharide A only in lacking 1 of the 2 pyruvate residues.