Natural killer (NK) cells from nonhuman primates have not been completely characterized, and methods for expanding nonhuman primates NK cells in vitro have been described only in rhesus species. The purpose of this report was to characterize NK cells in pigtail macaques (Macaca nemestrina), a species that is frequently used in studies of transplantation biology/immunology, virology, vaccine development, and reproductive biology. NK cells from Macaca nemestrina peripheral blood were best defined by the expression of CD16 and CD8α, and the absence of CD3. Subsets of these cells express CD56, NKp30, and NKp46. An enhanced ability to kill K562 cells was not present in fluorescence activated cell sorted (FACS)-purified CD16–/CD3+ and CD16–/CD56+ cells isolated from fresh peripheral blood. However, FACS-purified CD16+/CD3– and CD16+/CD56– cells were highly efficient killers of K562 cells. Macaca nemestrina NK cells can be expanded by in vitro culturing of FACS-purified CD16+/CD2–/CD3–/CD56– cells, or from peripheral blood cells depleted of cells expressing CD3, CD4, and HLA-DR. Cells in these cultures expand 70-fold after 21 days of culturing. After culturing, these cells express high levels of natural cytotoxicity receptors (NCRs) NKp30 and NKp46. NK cell populations obtained from FACS-purified CD16+/CD3–, CD16+/CD56– cells and CD3/CD4/HLA-DR-depleted cells were highly efficient killers of K562 cells. These data suggest that a population of highly enriched cytolytic NK cells can be obtained from purified CD16+/CD3– and CD16+/CD56– cells obtained from peripheral blood, as well as from cells that have been cultured and expanded from peripheral blood that is depleted of CD3/CD4/HLA-DR-expressing cells. Am. J. Primatol. 68:753–764, 2006. © 2006 Wiley-Liss, Inc.