The search for a novel producer of glycolipid biosurfactants, mannosylerythritol lipids (MELs), was undertaken on the basis of the analysis of ribosomal DNA sequences of yeast strains of the genus Pseudozyma. In the course of the investigation, Pseudozyma siamensis CBS 9960, which is closely related to Pseudozyma shanxiensis, a known MEL-C producer but with a different morphology, was found to accumulate a large amount of glycolipids. On thin layer chromatography, the extracellular glycolipids showed nearly the same spots as those of the MELs produced by P shanxiensis. However, the result of high-performance liquid chromatography analysis revealed that the present strain has a much higher glycolipid production yield than P shanxiensis. From the structural characterization by H-1 and C-13 NMR, the major glycolipid (more than 84% of the total) was identified as a mixture of 4-O-[(2',4'-di-O-acetyl-3'-O-alka(e)noyl)-beta-D-mannopyranosyl]-D-eryth ritol and 4-O-[(4'-O-acetyl-3'-O-alka(e)noyl-2'-O-butanoyl)-beta-D-mannopyranosyl] -D-erythritol, both of which are types of MEL-C. The present MEL-C possessed a short-chain acid (C-2 or C-4) at the C-2' position and a long-chain acid (C,,) at the C-3' position of the mannose moiety, and thus, the hydrophobic part was considerably different from that of conventional MELs, which mainly possess two medium-chain acids (C-10) at the C-2' and C-3' positions. Under optimal growth conditions with safflower oil in a shake culture, the total amount of MELs reached approximately 19 g/l after 9 d at 25 degrees C. We further investigated the interfacial properties of the present MEL-C, considering its unique hydrophobic structure. The observed critical micelle concentration (CMC) and the surface tension at the CMC of the MEL were 4.5x 10(-6) M and 30.7 mN/m, respectively. In addition, on a water penetration scan, the MEL efficiently formed the liquid crystal phases such as hexagonal (H) and lamella (L.) at a wide range of concentrations. These results demonstrated that the newly identified MEL-C produced by P siamensis exhibits not only high surface activity but also excellent self-assembling properties, and should facilitate the development of promising yeast biosurfactants.