The role of glycosphingolipids as adhesion receptors for yeasts was examined. Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae, as well as Histoplasma capsulatum and Sporotrichum schenckii (in their yeast phases), bound specifically to lactosylceramide (Gal beta 1-4Glc beta 1-1Cer), as measured by overlaying glycosphingolipid chromatograms with 125I-labeled organisms. An unsubstituted galactosyl residue was required for binding, because the yeasts did not bind to glucosylceramide (Glc beta 1-1Cer) derived from lactosylceramide by treatment with beta-galactosidase or to other neutral or acidic glycosphingolipids tested that contained internal lactosyl residues. Interestingly, the yeasts preferentially bound to the upper band of the lactosylceramide doublet in human lung and bovine erythrocytes, suggesting that the ceramide structure also affects binding. Active metabolism of the yeasts was required for binding to lactosylceramide, as binding was maximal in buffer containing glucose and was almost completely abolished in nutrient-deficient medium. C. neoformans also bound to human glioma brain cells grown in monolayers, and this binding was inhibited by liposomes containing lactosylceramide but not by liposomes containing glucosylceramide. Lactosylceramide is a major glycosphingolipid in these cells and the only one to which the yeasts bound. As lactosylceramide is widely distributed in epithelial tissues, this glycosphingolipid may be the receptor for yeast colonization and disseminated disease in humans.