Understanding of heterogeneous NK subsets is important for the study of NK cell biology and development, and for the application of NK cell-based therapies in the treatment of disease. Here we demonstrate that the surface expression of CD94 can distinctively divide mouse NK cells into two approximately even CD94(low) and CD94(high) subsets in all tested organs and tissues. The CD94(high) NK subset has significantly greater capacity to proliferate, produce IFN-gamma, and lyse target cells than does the CD94(low) subset. The CD94(high) subset has exclusive expression of NKG2A/C/E, higher expression of CD117 and CD69, and lower expression of Ly49D (activating) and Ly49G2 (inhibitory). In vivo, purified mouse CD94(low) NK cells become CD94(high) NK cells, but not vice versa. Collectively, our data suggest that CD94 is an Ag that can be used to identify functionally distinct NK cell subsets in mice and could also be relevant to late-stage mouse NK cell development.