Enterovirus 70 (EV70), the causative agent of acute hemorrhagic conjunctivitis, exhibits a restricted tropism for conjunctival and corneal cells in vivo but infects a wide spectrum of mammalian cells in culture. Previously, we demonstrated that human CD55 is a receptor for EV70 on HeLa cells but that EV70 also binds to sialic acid-containing receptors on a variety of other human cell lines. Virus recognition of sialic acid attached to underlying glycans by a particular glycosidic linkage may contribute to host range, tissue tropism, and pathogenesis. Therefore, we tested the possibility that EV70 binds to alpha2,3-linked sialic acid, like other viruses associated with ocular infections. Through the use of linkage-specific sialidases, sialyltransferases, and lectins, we show that EV70 recognizes alpha2,3-linked sialic acid on human corneal epithelial cells and on U-937 cells. Virus attachment to both cell lines is CD55 independent and sensitive to benzyl N-acetyl-alpha-D-galactosaminide, an inhibitor of O-linked glycosylation. Virus binding to corneal cells, but not U-937 cells, is inhibited by proteinase K, but not by phosphatidylinositol-specific phospholipase C treatment. These results are consistent with the idea that a major EV70 receptor on corneal epithelial cells is an O-glycosylated, non-glycosyl phosphatidylinositol-anchored membrane glycoprotein containing alpha2,3-linked sialic acid, while sialylated receptors on U-937 cells are not proteinaceous.