Mammalian glycolipid transfer proteins (GLTPs) facilitate the selective transfer of glycolipids between lipid vesicles in vitro. Recent structural determinations of the apo- and glycolipid-liganded forms of human GLTP have provided the first insights into the molecular architecture of the protein and its glycolipid binding site (Malinina, L., Malakhova, M. L., Brown, R. E., and Patel, D. J. (2004) Nature 430, 1048-1053). In the present study, we have evaluated the functional consequences of point mutation of the glycolipid liganding site of human GLTP within the context of a carrier-based mechanism of glycolipid intermembrane transfer. Different approaches were developed to rapidly and efficiently assess the uptake and release of glycolipid by GLTP. They included the use of glass-immobilized, glycolipid films to load GLTP with glycolipid and separation of GLTP/glycolipid complexes from vesicles containing glycolipid (galactosylceramide or lactosylceramide) or from monosialoganglioside dispersions by employing nickel-nitrilotriacetic acid-based affinity or gel filtration strategies. Point mutants of the sugar headgroup recognition center (Trp-96, Asp-48, Asn-52) and of the ceramide-accommodating hydrophobic tunnel (Phe-148, Phe-183, Leu-136) were analyzed for their ability to acquire and release glycolipid ligand. Two manifestations of point mutation within the liganding site were apparent: (i) impaired formation of the GLTP/glycolipid complex; (ii) impaired acquisition and release of bound glycolipid by GLTP. The results are consistent with a carrier-based mode of GLTP action to accomplish the intermembrane transfer of glycolipid. Also noteworthy was the inefficient release of glycolipid by wtGLTP into phosphatidylcholine acceptor vesicles, raising the possibility of a function other than intermembrane glycolipid transfer in vivo.