Melanomas are characterized by high metastatic potential, with regional lymph node (LN) representing the most frequent site of early dissemination in this disease. These regional LN also represent the primary site for differentiation of natural killer cells (NK cells). While blood-derived NK cells can efficiently lyse melanoma cells isolated from metastatic LN (M-LN), there has been no study of the properties of the most disease-relevant NK cells isolated from M-LN in melanoma patients. Here we report that M-LN contain 0.5-11% of CD56bright NK cells among CD45+ hematopoietic cells present and that this cell population surrounded tumor cell clusters in M-MN. This NK cell population was characterized by expression of CD62L, chemokine receptors and high levels of natural cytotoxicity receptors (NCRs), NKG2D and DNAM-1. Expression of NCR-NKp30 and NKG2D correlated negatively with percentages of tumor cells in M-LN. Interestingly, M-LN contained a unique subset of mature CD56brightCD16+ NK cells displaying co-regulated expression of NCR and NKG2D activating receptors. Ex vivo analyses suggested that M-LN-derived NK cells were inactive but could be activated by appropriate cytokine signals (IL-2 or IL-15), could lyse metastatic melanoma cells in a highly efficient manner compared to blood-derived NK cells. Taken together, the results offer evidence that adjuvant immunotherapy that targets NK cells in M-LN for activation may improve treatment of sentinel LN-positive melanoma patients.