The interactions of a succinoyl bacterial trehalose lipid biosurfactant produced by Rhodococcus sp. with phospholipid vesicles, leading to membrane permeabilization, are studied by means of calorimetric and fluorescence and absorption spectroscopical techniques in search for a molecular model. The critical micelle concentration (CMC) of trehalose lipid is determined, by surface tension measurements, to be 300 muM. Binding of trehalose lipid to palmitoyloleoylphosphatidylcholine membranes is studied by means of isothermal titration calorimetry. The partition constant, in conjunction with the CMC, indicates that trehalose lipid behaves as a weak detergent, which prefers membrane incorporation over micellization. Addition of trehalose lipid to palmitoyloleoylphosphatidylcholine large unilamellar vesicles results in a size-selective leakage of entrapped solutes to the external medium. Experimental evidence is provided to support the requirement of a stage of flip-flop prior to membrane permeabilization, and the rate of flip-flop is measured using fluorescent probes assays. The lipid composition of the target membrane is found to modulate the leakage process to a great extent. It is proposed that trehalose lipid incorporates into phosphatidylcholine membranes and segregates within lateral domains which may constitute membrane defects or "pores", through which the leakage of small solutes might take place. The results presented here contribute to the knowledge of the molecular mechanisms underlying the membrane-related biological actions of this bacterial trehalose lipid biosurfactant.