We have cloned Gb8 synthase, the key α1,4-galactosyl-transferase in globo-series glycosphingolipid (GSL) synthesis, via a phenotypic screen, which previously yielded iGb3 synthase, the α1,3-galactosyltransferase required in isoglobo-series GSL (Keusch, J. J., Manzella, S. M., Nyame, K. A., Cummings, R. D., and Baenziger, J. U. (2000) J. Biol. Chem. 33). Both transferases act on lactosylceramide, Galβ1,4Glcβ1Cer (LacCer), to produce Gb3 (Galα1,4LacCer) or iGb3 (Galα1,3LacCer), respectively. GalNAc can be added sequentially to either Gb3 or iGb3 yielding globoside and Forssman from Gb3, and isogloboside and isoForssman from iGb3. Gb3 synthase is not homologous to iGb3 synthase but shows 43% identity to a human α1,4GlcNAc transferase that transfers a UDP-sugar in an α1,4-linkage to a β-linked Gal found in mucin. Extensive homology (35% identity) is also present between Gb3 synthase and genes in Drosophila melanogaster and Arabidopsis thaliana, supporting conserved expression of an α1,4-glycosyltransferase, possibly Gb3 synthase, throughout evolution. The isolated Gb3 synthase cDNA encodes a type II transmembrane glycosyltransferase of 360 amino acids. The highest tissue expression of Gb3 synthase RNA is found in the kidney, mesenteric lymph node, spleen, and brain. Gb3 glycolipid, also called P(k) antigen or CD77, is a known receptor for veretoxins. CHO cells that do not express Gb3 and are resistant to verotoxin become susceptible to the toxin following transfection with Gb3 synthase cDNA.