A glucuronic acid-containing diacylglycerol was isolated from exponentially growing Mycobacterium smegmatis. Structural analysis of the purified glycolipid, performed by gas chromatography-mass spectrometry, fast atom bombardment-mass spectrometry, and high resolution proton NMR, indicated the structure 3-(O-alpha-D-glucuronopyranosyl)-1,2-diacyl-sn-glycerol. Two forms of the glycolipid were observed differing in fatty acid composition. Both molecular species contained a hexadecanoic acid residue, whereas the second acyl group was either tuberculostearic acid (10-methylstearic acid) or octadecenoic acid. The inherent antigenicity of the glycolipid was shown by its ability to bind to anti-Mycobacterium avium (serovar 26) and anti-Mycobacterium tuberculosis sera by Western blot-type thin-layer chromatography. This is the second instance of the isolation of a glycosyl diacylglycerol from members of the Mycobacterium genus, further confirming its close relationship to Gram-positive bacteria.