NKG2D serves as one of the most potent activating receptors for effector lymphocytes in peripheral tissues. Here we report the characterization of Letal, the first human transmembrane NKG2D ligand lacking an immunoglobulin-like a-3 ectodomain. Letal is constitutively expressed by a variety of normal tissues, and is up-regulated in tumor cells of different origins. Unlike other NKG2D ligands, Letal mRNA expression progressively decreased after treatment of tumor cells with retinoic acid. Simultaneous T-cell receptor activation and engagement of Letal stimulated proliferation of CD8+ cells and dramatically increased IL-2 and IFN-g secretion. In addition, Letal induced the killing of cancer cells by CD8+ and NK cells. These results suggest that Letal delivers activating signals to NK cells and promotes tumor immune surveillance by inducing the expansion of anti-tumor cytotoxic lymphocytes.