Carbohydrates on tumor cells have been shown to play an important role in tumor metastasis. We demonstrated before that CD24, a M_r 35,000–60,000 mucine-type glycosylphosphatidylinositol-linked cell surface molecule, can function as ligand for P-selectin and that the sialylLe^xcarbohydrate is essential for CD24-mediated rolling of tumor cells on P-selectin. To investigate the role of both antigens more closely, we transfected human A125 adenocarcinoma cells with CD24 and/or fucosyltransferase VII (Fuc TVII) cDNAs. Stable transfectants expressed CD24 and/or sialylLe^x. Biochemical analysis confirmed that in A125-CD24/FucTVII double transfectants, CD24 was modified with sialylLe^x. Only double transfectants showed rolling on P-selectin in vivo. When injected into mice, double transfectants arrested in the lungs, and this step was P-selectin dependent because it was strongly enhanced in lipopolysaccharide(LPS) pretreated wild-type mice but not in P-selectin knockout mice. CD24 modified by sialylLe^x was required on the tumor cells because the LPS-induced lung arrest was abolished by removal of CD24 from the cell surface by phosphatidylinositol-specific phospholipase C. A125-FucTVII single transfectants expressing sialylLe^x but not CD24 did not show P-selectin-mediated lung arrest. The sialylLe^x epitope is abundantly expressed on human carcinomas, and significant correlations between sialylLe^x expression and clinical prognosis exist. Our data suggest an important role for sialylLe^x-modified CD24 in the lung colonization of human tumors.