Natural killer (NK) cells in rhesus macaques have been variably defined as CD3- CD16+ or CD3- CD8+, although only limited efforts have been made to validate these definitions rigorously. To better understand the role of NK cells in macaque disease models, we undertook a multiparameter analysis of macaque NK cells employing four-colour flow cytometry and a panel of lineage-specific and non-lineage-specific lymphocyte markers. Using this approach, we identified two distinct populations of candidate NK cells: a major CD8bright CD16+ population and a minor CD8bright CD16- population. Further analysis of the major and minor NK cell populations revealed the expression of multiple markers characteristic of NK cells, including CD2, CD7, CD16, CD161, NKG2A and granzyme B. In addition, a CD56+ subset of cells within the minor rhesus NK population was identified which expressed chemokine and lymph node homing receptors similar to those expressed by the CD56bright NK cell population identified in humans. Cytolytic assays confirmed that the phenotypically defined rhesus NK cells lysed NK-susceptible target cells. Our observations support the existence of several distinct subpopulations of rhesus macaque NK cells, which have significant phenotypic and functional similarities to their human counterparts. These improved immunophenotypic definitions of macaque NK cells should facilitate future analysis of innate immune responses in rhesus macaques and the role of NK cells in AIDS pathogenesis in Simian immunodeficiency virus (SIV)-infected macaques.