Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes by Rhodococcus sp. These compounds show not only unique interfacial properties but also versatile biochemical actions. In this study, we determined for the first time the complete structure of the main component, namely STL-1, based on NMR, MALDI-TOF/MS and GC-MS analyses. The present STL-1 produced from n-hexadecane was identified as 3,4-di-O-alkanoyl-2-O-succinoyl-alpha-D-glucopyranosyl-2'-O-succinoyl-alpha-D-glucopyranoside. The major fatty acid of STL-1 was C(16), indicating that n-hexadecane as the carbon source is directly incorporated into the carbohydrate moiety via terminal oxidation. We also investigated its surface active properties using a Whilhelmy method at 25 degrees C. The estimated CMC and gamma(CMC) values for STL-1 were 5.6x10(-6) M and 19.0 mN/m, and those for sodium salt (NaSTL-1) were 7.7x10(-6) M and 23.7 mN/m, respectively. The high activity of STL-1 was probably due to the unique molecular structure resulting from its multiple functional groups including succinic acids and two long fatty acids. Accordingly, STL-1 and its sodium salt were demonstrated to exhibit an excellent surface-activity at remarkably low concentrations, and should have great potential for environmentally friendly surfactants.