Expression of sialyl Lewisx (sLex) and sLea on tumor cells is thought to facilitate metastasis by promoting cell adhesion to selectins on vascular endothelial cells. Experiments supporting this concept usually bypass the early steps of the metastatic process by employing tumor cells that are injected directly into the blood. We investigated the relative role of sLex oligosaccharide in the dissemination of breast carcinoma, employing a spontaneous murine metastasis model. An sLex deficient subpopulation of the 4T1 mammary carcinoma cell line was produced by negative selection using the sLex-reactive KM93 MAb. This subpopulation was negative for E-selectin binding but retained P-selectin binding. Both sLex-negative and -positive cells grew at the same rate; however, sLex-negative cells spread more efficiently on plates and had greater motility in wound-scratch assays. Mice inoculated in the mammary fat pad with sLex-negative and -positive variants produced lung metastases. However, the number of lung metastases was significantly increased in the group inoculated with the sLex-negative variant (p = 0.0031), indicating that negative selection for the sLex epitope resulted in enrichment for a subpopulation of cells with a high metastatic phenotype. Cell variants demonstrated significant differences in cellular morphology and pattern of tumor growth in primary and secondary tumor sites. These results strongly suggest that loss of sLex may facilitate the metastatic process by contributing to escape from the primary tumor mass. © 2005 Wiley-Liss, Inc.