Melanoma has a high tendency to metastasize to lymph nodes, which is one of the clinicopathological factors to indicate poor prognosis. Recent investigations have shown the importance of lymphangiogenesis in lymph node metastasis in a variety of human tumors including melanoma. However, molecular mechanism of lymphatic metastasis is still poorly defined. We examined influence of interactions between normal lymphatic endothelial cells (LECs) and melanoma cells on cell migration. Medium conditioned with LEC (LEC-CM) contained chemotactic and chemokinetic activities for human melanoma cell lines. The chemotactic activity was fractionated in more than 100 kDa, and inactivated by heat-treatment. The chemotactic activity of LEC-CM was abolished by immunodepletion with anti-laminin-1 antibody. And immunoprecipitation and Western blot analyses revealed that LEC-CM contained laminin-421. When melanoma C8161 cells were treated with function-blocking antibodies to integrin α3 or α6, their chemotactic responses to LEC-CM were markedly reduced. Furthermore, the knock-down of tetraspanin CD151 weakened the chemotactic responses of C8161 and MeWo cells to LEC-CM. These data suggest that laminin-421 secreted by LEC possibly facilitates lymphatic metastasis through the induction of chemotaxis of melanoma cells.