The bacterium Tsukamurella sp. nov., isolated from soil, was found to produce novel glycolipids when grown on sunflower oil as the sole carbon source. The glycolipids were isolated by chromatography on silica columns and their structures elucidated using a combination of multidimensional NMR and MS techniques. The three main components are 2,3-di-O-acyl-alpha-D-glucopyranosyl-(1-1)-alpha-D-glucopyranose, 2,3-di-O-acyl-beta-D-glucopyranosyl-(1-2)-4,6-di-O-acyl-alpha-D-glucopyranosyl-(1-1)-alpha-D-glucopyranose and 2,3-di-O-acyl-beta-D-glucopyranosyl-(1-2)-beta-D-galactopyranosy-(1-6)-4,6-di O-acyl-alpha-D-glucopyranosyl-(1-1)-alpha-D-glucopyranosl which are linked to fatty acids varying in chain length from C-4 to C-18. The glycolipids are mainly extracellular but are also found attached to the cell walls. During the cultivation the composition of the glycolipids changed from disaccharide- to tri- and tetrasaccharide lipids. The glycolipids show good surface-active behaviour and have antimicrobial properties.