The effect of cholesterol on the vesicle formation of a rhamnolipid biosurfactant extracted from the liquid culture of Pseudomonas aeruginosa SP4 was investigated. The rhamnolipid vesicles were prepared in a phosphate-buffer saline (PBS) solution (pH 7.4) at a biosurfactant concentration of 2.6 mM, or 6.5 times the critical micelle concentration (CMC), with various amounts of cholesterol. The biosurfactant solution was characterized using turbidity, zeta potential, and dynamic light scattering (DLS) measurements. The morphology of the rhamnolipid vesicles formed at different cholesterol concentrations was examined with the use of transmission electron microscopy (TEM). The results showed that the rhamnolipid biosurfactant formed spherical vesicles both in the absence and presence of cholesterol, but the incorporation of the cholesterol into the bilayer membrane reduced the vesicle size. Sudan III, a water-insoluble dye, was used as a model hydrophobic compound in the encapsulation experiment. The encapsulation efficiency (E%) of the rhamnolipid vesicles was affected by the cholesterol concentration and the initial Sudan III concentration. The maximum E% of nearly 90% was achieved at the cholesterol concentration of 100 mu M and the initial Sudan III concentration of 8.8 mu M. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.