Phospholipid exchange protein from beef heart or beef liver does not catalyze the transfer of phosphatidylcholine from multilamellar vesicles of phosphatidylcholine. Certain combinations of phospholipids, however, do yield multilamellar vesicles that will exchange phosphatidylcholine with liposomes in the presence of exchange protein. Multilamellar vesicles of phosphatidylcholine:phosphatidylethanolamine:cardiolipin (70:25:5, mol%) can be used in place of mitochondria or erythrocyte ghosts as an improved acceptor particle in the study of liposome structure with phospholipid exchange proteins. These multilamellar vesicles act as a well-defined reservoir of unlabeled phosphatidylcholine with 7% exchangable phospholipid. When the distribution of phosphatidylcholine in liposomes is studied by the exchange protein technique, results can be influence by the choice of phospholipid acceptor particle. With mitochondria as acceptor particle, the percentage of phosphatidylcholine in the outer monolayer of a liposome appears to be 60%, whereas a value of 70% is obtained when multilamellar vesicles are the acceptor. The discrepancy can be explained by a heterogeneity in liposomes prepared by sonication. A size-dependent fusion or adsorption process occurs between liposomes and mitochondria; the very small liposomal vesicles, obtained by gel filtration, combine nearly quantitatively with the natural membrane. This phenomenon is not seen with multilamellar vesicles. Thus by using multilamellar vesicles one obtains a less biased estimate of phospholipid distribution between inner and outer layers of liposomes.