Breast cancer often spreads from the primary tumor to regional lymph nodes. Lymph node status provides clinically important information for making treatment decisions. Spread via lymphatics is also important for the biology of breast cancer, as tumor cells in lymph nodes may provide a reservoir of cells leading to distant, lethal metastases. Improved understanding of the biology of lymphatic spread thus is important for improved breast cancer survival. Advances towards understanding the interactions between tumors cells and lymphatic vessels have in part been limited by the lack of suitable cell lines and experimental models. We have addressed this need by developing a new model of lymphatic metastasis. Here we describe the establishment of 468LN cells, a variant of the MDA-MB-468 human breast adenocarcinoma cell line, which produces extensive lymph node metastasis following orthotopic injection of nude mice. 468LN cells are also more aggressive in vitro, produce more osteopontin and express different surface integrins compared to the parent line. The dramatic in vitro and in vivo phenotypic and molecular differences of 468LN and parental 468GFP cells make this pair of cell lines a unique model for the specific study of lymph node metastasis of breast cancer.