Hepatocyte growth factor (HGF) is a heparin-binding pleiotropic factor that acts on a variety of epithelial cells. The interaction of human HGF with glycolipids was studied by overlaying them with 125I-HGF on thin layer chromatograms and by a solid-phase assay using lipids adsorbed on microtiter plates. Among various glycolipids tested, HGF was found to bind to sulfoglycolipids, including galactosylceramide sulfate (SM4), lactosylceramide sulfate (SM3), and gangliotriaosylceramide bis-sulfate. In contrast, HGF failed to bind to gangliosides or neutral glycolipids. HGF binding to SM4 was strongly inhibited by dextran sulfate, heparin, and fucoidan, whereas neither keratan sulfate nor hyaluronic acid had any inhibitory activity. When glycolipids from a renal cancer cell line, SMKT-R3, which overexpresses sulfoglycolipids, were developed on a thin layer chromatogram, SM4 and SM3 were the only glycolipids that bound HGF. We further examined the effect of the incorporation of glycolipids into SMKT-R3 cells on HGF binding to the cells. The incorporation of SM4 into the cells enhanced HGF binding to SMKT-R3 cells, while that of galactosylceramide, a precursor of SM4, had no effect. These observations indicated that SM4 exogenously incorporated into the cell membranes could react with HGF and suggested that endogenous sulfoglycolipids on SMKT-R3 cells might function as reservoirs for HGF.