The bichromophoric and monochromophoric probes, 1,32-dihydroxy-dotriacontane-bis(Rhodamine) 101 ester (Rh101C32Rh101) and Rhodamine 101 octadecyl ester (Rh101C18), respectively, were solubilised in unilamellar lipid vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). At molar ratios of less than 1/5000 for Rh101C18/lipid and Rh101C32Rh101/lipid, intermolecular electronic energy migration is negligible, contrary to the intramolecular donor-donor energy migration (DDEM) within the Rh101C32Rh101 molecules. The time-resolved fluorescence anisotropy determined for Rh101C18and Rh101C32Rh101 were globally analysed by using a recently developed model that accounts for reorientational motions, as well as the rate of DDEM (L. B-Å. Johansson, F. Bergström, P. Edman, I. V. Grechishnikova and J. G. Molotkovsky, J. Chem. Soc., Faraday Trans., 1996, 92, 1563). It was found that the rate of intramolecular DDEM within Rh101C32Rh101 is typically about 3 × 109s-1. In vesicles formed by DOPC and POPC, the distance between the Rhodamine groups of Rh101C32Rh101 is found to be about 35 Å, while it is about 32 Å in DPPC vesicles. These values are in excellent agreement with reported thicknesses of the lipid bilayers. Thus, the present work strongly suggests that the Rh101C32Rh101 molecules are spanning across the lipid bilayer, that is, the Rhodamine residues are located on opposite sides of the lipid bilayer.