Galectin-4, a member of the galectin family, is expressed in the epithelium of the alimentary tract. It has two tandemly repeated carbohydrate recognition domains and specifically binds to an SO3- -->3Galbeta1-->3GalNAc pyranoside with high affinity (Ideo, H., Seko, A., Ohkura, T., Matta, K. L., and Yamashita, K. (2002) Glycobiology 12, 199-208). In this study, we found that galectin-4 binds to glycosphingolipids carrying 3-O-sulfated Gal residues, such as SB1a, SM3, SM4s, SB2, SM2a, and GM1, but not to glycosphingolipids with 3-O-sialylated Gal, such as sLc4Cer, snLc4Cer, GM3, GM2, and GM4, using both an enzyme-linked immunosorbent assay and a surface plasmon resonance assay. A confocal immunocytochemical assay showed that galectin-4 was colocalized with SB1a, GM1, and carcinoembryonic antigen (CEA) in the patches on the cell surface of human colon adenocarcinoma CCK-81 and LS174T cells. This localization was distinct from caveolin/VIP21 localization. Furthermore, immobilized galectin-4 promoted adhesion of CCK-81 cells through the sulfated glycosphingolipid, SB1a. CEA also bound to galectin-4 with KD value of 2 x 10(-8) m by surface plasmon resonance and coimmunoprecipitated with galectin-4 in LS174T cell lysates. These findings suggest that SB1a and CEA in the patches on the cell surface of human colon adenocarcinoma cells could be biologically important ligands for galectin-4.