Rhamnolipids are biosurfactants produced by Pseudomonas aeruginosa which are well known for their potential industrial and environmental uses. Rhamnolipids have gained considerable interest in recent years due to their potential use in cosmetics and pharmaceutics. They also show broad biological activities and have potential applications as therapeutic agents. The amphiphilic nature of rhamnolipids points to the membrane as their hypothetical site of action. We have purified dirhamnolipid and studied its interaction with phosphatidylcholine membranes, using differential scanning calorimetry, X-ray diffraction and infrared spectroscopy. It has been found that dirhamnolipid greatly affects the gel to liquid crystalline phase transition of phosphatidylcholines, broadening and shifting the transition to lower temperatures. Dirhamnolipid increases the interlamellar repeat distance of phosphatidylcholines and reduces the long-range order of the multilamellar systems. The phospholipid hydrocarbon chain conformational disorder is increased and the packing of the phospholipid molecules is perturbed in the presence of dirhamnolipid. The above evidence supports the idea that dirhamnolipid intercalates into the phosphatidylcholine bilayers and produces structural perturbations which might affect the function of the membrane.