Rat liver cytosol fraction facilitated the transfer of [3H]glucosylceramide and [3H]-galactosylceramide from donor to acceptor liposomes, which contained one of these glycolipids in phospholipid-cholesterol. Lactosylceramide transfer activity was not detectable in the cytosol fraction. On Sephadex G-75 gel filtration of the liver cytosol fraction, both the glucosylceramide and galactosylceramide transfer activities were eluted at an identical position with a Kav value of 0.364, which corresponds to a molecular weight of 19,500; a little lactosylceramide transfer activity was found in the effluents containing the glycolipid transfer activities. The active protein fraction obtained by Sephadex G-75 chromatography of rat liver cytosol accelerated the transfer of galactosylceramide, glucosylceramide and lactosylceramide at relative rates of 100, 79, and 31, respectively. The rates of galactosylceramide and glucosylceramide transfer facilitated by the rat liver Sephadex G-75 fraction were not affected by either galactosylceramide, glucosylceramide, or lactosylceramide contained in the acceptor liposomes; acceptor liposomes lacking glycolipids acted efficiently as acceptors in the transfer reactions. For the lactosylceramide transfer reaction, significantly lower activity was detected when the acceptor liposomes contained either galactosylceramide or glucosylceramide. The translocation of [3H]-galactosylceramide from the donor to acceptor liposomes was confirmed by TLC and fluorography of the lipids extracted from the acceptor liposomes, which were separated from the donor liposomes after the incubation.