We have recently shown that interleukin-2 (IL-2)-activated CD8⁺CD44^hi cells from normal mice express both adaptive and innate immune system receptors and specifically kill syngeneic tumor cells, particularly those that express NKG2D ligands. Here we show that CD8⁺ T cells from antigen-expressing H-Y T-cell receptor (TCR) transgenic mice also exhibit characteristics of both T cells and natural killer (NK) cells. Interaction with cognate self-antigen was required for the optimal expansion of these cells in peripheral lymphoid tissues. Although these cells possess a higher activation threshold relative to naive T cells, they can be activated by cytokine alone in vitro. They also undergo bystander proliferation in response to a bacterial infection in vivo. Interestingly, upon activation, the cells express the NKG2D receptor as well as the DNAX activation protein 12 (DAP12) adaptor protein. We provide evidence that NKG2D can act additively with the TCR in the killing of target cells, and it can also function as a directly activating receptor in non-major histocompatibility complex (MHC)-restricted killing of target cells. These properties of CD8⁺ T cells from H-Y TCR transgenic mice are remarkably similar to CD8⁺CD44^hi cells that are found in normal mice. The H-Y TCR transgenic mice provide a well-defined system for characterizing the developmental biology and function of these cells. (Blood. 2004;104:2116-2123)