The kinetics of cholesterol and cholesterol sulfate (CS) movement between vesicles have been investigated. CS is widely distributed in cell membranes, plasma and skin and is similar in structure to cholesterol, but possesses an ionizable sulfate moiety at the 3 beta-position which imparts a negative charge at physiological pHs. Donor vesicles of various sizes ranging from 40 to 250 nm, composed of egg phosphatidylcholine (EPC)/sterol/N-palmitoyldihydrolactosylcerebroside (75:10:15 mole ratio) containing trace amounts of [3H]sterol, were used to monitor sterol transfer into a 10-fold excess of large unilamellar vesicles (LUV) composed of EPC with a diameter of 100 nm. The two populations of vesicles were separated by centrifugation following the addition of a lectin which caused the aggregation of donor vesicles. Both cholesterol and CS exhibited biphasic kinetics of exchange. The rate constants for efflux and transbilayer diffusion for both sterol molecules were determined after fitting kinetic data, using numerical integration, to a three-compartment model, which includes the inner and outer monolayers of donor vesicles and the acceptor bilayer. The rate of intermembrane exchange for CS was approximately 10-fold faster than for cholesterol in all liposomes tested. Using the kinetic model, a rate of transbilayer movement for cholesterol and CS was estimated. In both cases, it was found to be slower than the rate of efflux from the surface of vesicles. For vesicles containing CS, the surface charge was monitored to demonstrate that the slowly exchanging pool was located in the inner monolayer, and the rapidly exchanging pool in the outer half of the bilayer. For cholesterol, it was not possible to distinguish between this model and one where lateral domains of cholesterol within the plane of the bilayer may influence the kinetics of exchange.