FTIR spectroscopy and fluorescence polarization were used to show that a bacterial dirhamnolipid interacts with phospholipid membranes composed of DPPC, altering both the acyl chain and the interfacial region of the bilayer. Incorporation of increasing amounts of dirhamnolipid into (2)H-DPPC membranes broadened the transition and shifted the transition temperature toward lower values, according to the effect on the CD(2) stretching vibration. Examination of the (13)C=O stretching band of (13)C-DPPC indicated that, both below and above the phase transition, dirhamnolipid produced a shift of the band frequency toward higher values, indicating a strong dehydration of the phospholipid C=O groups, and therefore of the interfacial region of the membrane. The effects on DPH and TMA-DPH fluorescence polarization provided additional support to hypothesize on the location of trehalose lipid within the bilayer. The results shown here could help to explain some of the interesting membrane-related biological actions of rhamnolipids reported before.