A biosurfactant-producing strain S6 was isolated from oil-containing wastewater and identified as Pseudomonas aeruginosa based on physiological and biochemical tests together with 165 rDNA sequence analysis. Thin layer chromatography (TLC) and high-performance liquid chromatography electrospray ionization mass spectra (HPLC-ESI-MS) worked together to reveal that the strain S6 produced rhamnolipid biosurfactant. Mass spectrometry confirmed the presence of some major components in the rhamnolipid surfactant showing m/z of 675.8, 529.6, 503.3 and 475.4, which corresponded to RhaRhaC(10)C(12:1), RhaC(12:1)C(10), RhaC(10)C(10) and RhaC(8)C(10), respectively. The biosurfactant produced by strain S6 had the ability to decrease the surface tension of water from 72 to 33.9 mN m(-1), with the critical micelle concentration (CMC) of 50 mg L(-1). Emulsification experiment indicated that this biosurfactant effectively emulsified the crude petroleum and the measurements of surface tension demonstrated that the biosurfactant possessed stable surface activity at variable ranges of pH and salinity. The biosurfactant also exhibited good performance of phenanthrene solubilization with about 23 times higher solubility of phenanthrene in water than the control. Thus, this biosurfactant may have a potential for application in bioremediation of crude oil contamination. (C) 2008 Elsevier Ltd. All rights reserved.