The transfer kinetics of the negatively charged glycosphingolipid II3-N-acetylneuraminosyl-gangliotetraosylceramide (GM1) were investigated by monitoring tritiated GM1 movement between donor and acceptor vesicles. After appropriate incubation times at 45 degrees C, donor and acceptor vesicles were separated by molecular sieve chromatography. Donors were small unilamellar vesicles produced by sonication, whereas acceptors were large unilamellar vesicles produced by either fusion or ethanol injection. Initial GM1 transfer to acceptors followed first-order kinetics with a half-time of about 40 h assuming that GM1 is present in equal mole fractions in the exterior and interior surfaces of the donor vesicle bilayer and that no glycolipid flip-flop occurs. GM1 net transfer was calculated relative to that of [14C]cholesteryl oleate, which served as a nontransferable marker in the donor vesicles. Factors affecting the GM1 interbilayer transfer rate included phospholipid matrix composition, initial GM1 concentration in donor vesicles, and the GM1 distribution in donor vesicles with respect to total lipid symmetry. The findings provide evidence that GM1 is molecularly dispersed at low concentrations within liquid-crystalline phospholipid bilayers.