Abstract Ly49E is an unusual member of the Ly49 family that is expressed on fetal NK cells, epithelial T cells, and NKT cells, but not on resting adult NK cells. Ly49Ebgeo/bgeo mice in which the Ly49E gene was disrupted by inserting a β-geo transgene were healthy, fertile, and had normal numbers of NK and T cells in all organs examined. Their NK cells displayed normal expression of Ly49 and other NK cell receptors, killed tumor and MHC class I-deficient cells efficiently, and produced normal levels of IFN-γ. In heterozygous Ly49E+/bgeo mice, the proportion of epidermal T cells, NKT cells, and IL-2–activated NK cells that expressed Ly49E was about half that found in wild-type mice. Surprisingly, although splenic T cells rarely expressed Ly49E, IL-2–activated splenic T cells from Ly49Ebgeo/bgeo mice were as resistant to growth in G418 as NK cells and expressed similar levels of β-geo transcripts, suggesting that disruption of the Ly49E locus had increased its expression in these cells to the same level as that in NK cells. Importantly, however, the proportion of G418-resistant heterozygous Ly49E+/bgeo cells that expressed Ly49E from the wild-type allele was similar to that observed in control cells. Collectively, these findings demonstrate that Ly49E is not required for the development or homeostasis of NK and T cell populations or for the acquisition of functional competence in NK cells and provide compelling evidence that Ly49E is expressed in a probabilistic manner in adult NK cells and T cells.