An assay was developed to measure and directly compare transport of doxorubicin across right-side-out cytoplasmic membrane vesicles (ROV) and across model membranes (LUVET) composed of pure phospholipids, isolated from the corresponding cells. Escherichia coli was used as a model organism, since mutants are available which differ in phospholipid composition. Both in LUVET and ROV only passive diffusion across the bilayer is involved, because effects of drug concentration, pH, divalent cations, the phospholipid composition, and the active transport inhibitor verapamil were comparable. Permeability coefficients were about 2-3-times higher in ROV compared to LUVET. Furthermore, in LUVET an average activation energy of 87 kJ/mol and in ROV of 50 kJ/mol was observed. These differences are suggested to result from differences in membrane order between LUVET and ROV and differences in the temperature dependence of membrane order in LUVET and ROV, respectively. Because no background carrier-facilitated doxorubicin transport seems to be present, ROV are an excellent model system to study the effect of phospholipid composition on drug transport after expression of a multidrug resistance-conferring protein. Furthermore, data of passive diffusion of doxorubicin obtained with LUVET are representative for more complex, biologically relevant membrane systems.