Melanomas are characterized by high metastatic potential, with regional lymph node representing the most frequent site of early dissemination in this disease. These regional lymph nodes also represent the primary site for differentiation of natural killer (NK) cells. Although blood-derived NK cells can efficiently lyse melanoma cells isolated from metastatic lymph node (M-LN), there has been no study of the properties of the most disease-relevant NK cells isolated from M-LN in patients with melanoma. Here, we report that M-LN contains 0.5% to 11% of CD56^bright NK cells among CD45⁺ hematopoietic cells present and that this cell population surrounds tumor cell clusters in M-LN. This NK cell population was characterized by expression of CD62L, chemokine receptors, and high levels of natural cytotoxicity receptors (NCR), NK group 2 D (NKG2D), and DNAX accessory molecule 1 (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 CD56^brightCD16⁺ NK cells displaying coregulated 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 [interleukin (IL)-2 or IL-15], and could lyse metastatic melanoma cells in a highly efficient manner compared with 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 patients with sentinel lymph node–positive melanoma. Cancer Res; 74(1); 81–92. ©2013 AACR.