Human natural killer (NK) cells are one major component of lymphocytes that mediate early protection against viruses and tumor cells, and play an important role in immune regulatory functions. In this study, we demonstrated that human NK cells could be divided into four subsets, CD56hiCD16−, CD56loCD16−, CD56+CD16+ and CD56−CD16+, based on the expression of cell surface CD56 and CD16 molecules. Phenotypic analysis of NK cell subsets indicated that the expression of activation markers, adhesion molecules, memory cell markers, inhibitory and activating receptors, and intracellular proteins (granzyme B and perforin) were heterogeneous. Following interleukin (IL)-2 stimulation, interferon-γ was preferentially produced by CD56+CD16− NK cells and this subset showed more proliferative capacity. The cytolytic activity of both CD56+CD16− and CD56+/−CD16+ subsets could be augmented in response to IL-2. The data provided a new definition for NK cell subsets demonstrating their phenotypic and functional diversity and possible stage of NK cell differentiation in peripheral blood.