Natural killer (NK) cells and NK cell activities in the rhesus macaque have been incompletely characterized. Using a recently developed rhesus NK target cell line with down-regulated MHC-I (B116Lo) as stimulators and FACS-sorted cells as effectors in a 4-h [51Cr]-release assay we showed that the CD3-CD8lo subpopulation is the primary effector population for NK cell-mediated cytolysis. The majority of these cells co-express CD16, CD11b, NKG2D, and NKp46. To evaluate functional activity at the individual cell level, we employed intracellular cytokine staining and a flow cytometric assay for degranulation, based on cell surface CD107a expression. Flow cytometric analysis revealed that a greater proportion of NK cells degranulated than produced cytokines in response to B116Lo stimulation; the frequency of CD107a-expressing cells within the total NK cell population ranging from 5 to 39%. Somewhat surprisingly, we did not find a significant correlation between lysis, measured by [51Cr]-release assay, and the size of the degranulating NK cell population, implying that additional mechanisms may regulate lytic activity. Use of these approaches should facilitate an improved understanding of NK activity in the rhesus macaque.