Escherichia coli O157:H7 and other Shiga toxin (Stx)-producing E. coli (STEC) bacteria are not enteroinvasive but can cause hemorrhagic colitis. In some STEC-infected individuals, a life-threatening sequela of infection called the hemolytic uremic syndrome may develop that can lead to kidney failure. This syndrome is linked to the production of Stx by the infecting organism. For Stx to reach the kidney, the toxin must first penetrate the colonic epithelial barrier. However, the Stx receptor, globotriaosylceramide (Gb3), has been thought to be absent from human intestinal epithelial cells. Thus, the mechanisms by which the toxin associates with and traverses through the intestine en route to the kidneys have been puzzling aspects of STEC pathogenesis. In this study, we initially determined that both types of Stx made by STEC, Stx1 and Stx2, do in fact bind to colonic epithelia in fresh tissue sections and to a colonic epithelial cell line (HCT-8). We also discovered that globotetraosylceramide (Gb4), a lower-affinity toxin receptor derived from Gb3, is readily detectable on the surfaces of human colonic tissue sections and HCT-8 cells. Furthermore, we found that Gb3 is present on a fraction of HCT-8 cells, where it presumably functions to bind and internalize Stx1 and Stx2. In addition, we established by quantitative real-time PCR (qRT-PCR) that both fresh colonic epithelial sections and HCT-8 cells express Gb3 synthase mRNA. Taken together, our data suggest that Gb3 may be present in small quantities in human colonic epithelia, where it may compete for Stx binding with the more abundantly expressed glycosphingolipid Gb4.