The metastatic breast cancer cell line, 4T1, abundantly expresses the oligosaccharide sialylated Lewis x (sLex). SLex oligosaccharide on tumor cells can be recognized by E- and P-selectin, contributing to tumor metastatic process. We observed that both selectins reacted with this cell line. However, contrary to the E-selectin reactivity, which was sLex dependent, P-selectin reactivity with this cell line was sLex-independent. The sLex-Neg variant of the 4T1 cell line with markedly diminished expression of sLex and lack of sLea, provided a unique opportunity to characterize P-selectin ligands and their contribution to metastasis in the absence of overlapping selectin ligands and E-selectin binding. We observed that P-selectin binding was Ca2+-independent and sulfation-dependent. We found that P-selectin reacted primarily with cell surface chondroitin sulfate (CS) proteoglycans, which were abundantly and stably expressed on the surface of the 4T1 cell line. P-selectin binding to the 4T1 cells was inhibited by heparin and CS glycosaminoglycans (GAGs). Moreover, Heparin administration significantly inhibited experimental lung metastasis. In addition, the data suggest that surface CS GAG chains were involved in P-selectin mediated adhesion of the 4T1 cells to murine platelets and human umbilical vein endothelial cells. The data suggest that CS GAGs are also the major P-selectin-reactive ligands on the surface of human MDA-MET cells. The results warrant conducting clinical studies on the involvement of cell surface CS chains in breast cancer metastasis and evaluation of various CS types and their biosynthetic pathways as target for development of treatment strategies for antimetastatic therapy of this disease. © 2006 Wiley-Liss, Inc.