P‐selectin mediates rolling of neutrophils and other leukocytes on activated endothelial cells and platelets through binding to P‐selectin glycoprotein ligand‐1 (PSGL‐1). Certain PSGL‐1 negative tumor cell lines can bind P‐selectin under static conditions through the GPI‐linked surface mucin, CD24, but the physiological significance of this interaction and whether it can occur under flow conditions is not known. Here, we show that CD24⁺ PSGL‐1^– KS breast carcinoma cells attach to and roll on recombinant P‐selectin under a continuous wall shear stress, although at a lower density and higher velocity than CD24⁺ PSGL‐1⁺ cells, such as HL‐60. Adding excess soluble CD24 or removing CD24 from the cell surface with phosphatidylinositol‐phospholipase C (PI‐PLC) significantly reduced KS cell rolling on P‐selectin. The ability of KS cells to roll on P‐selectin was positively correlated with the CD24 expression level. Comparison with three other CD24⁺ cell lines established that expression of sialyl‐Lewis^x antigen was also necessary for CD24‐mediated rolling on P‐selectin. CD24 purified from KS cells supported rolling of P‐selectin transfectants, but not L‐selectin transfectants. Finally, KS cells rolled on vascular endothelium in vivo in a P‐selectin‐dependent manner. Together our data show that CD24 serves as a ligand for P‐selectin under physiological flow conditions. Interaction of tumor cells with P‐selectin via CD24 may be an important adhesion pathway in cancer metastasis.—Aigner, S., Ramos, C. L., Hafezi‐Moghadam, A., Lawrence, M. B., Friederichs, J., Altevogt, P., and Ley, K. CD24 mediates rolling of breast carcinoma cells on P‐selectin. FASEB J. 12, 1241–1251 (1998)