Natural killer (NK) cells attack tumor and infected cells, but the receptors and ligands that stimulate them are poorly understood. Here we report the expression cloning of two murine ligands for the lectin-like receptor NKG2D. The two ligands, H-60 and Rae1β, are distant relatives of major histocompatibility complex class I molecules. NKG2D ligands are not expressed by most normal cells but are up-regulated on numerous tumor cells. We show that mouse NKG2D is expressed by NK cells, activated CD8+ T cells and activated macrophages. Expression of either NKG2D ligand by target cells triggers NK cell cytotoxicity and interferon-γ secretion by NK cells, as well as nitric oxide release and tumor necrosis factor α transcription by macrophages. Thus, through their interaction with NKG2D, H-60 and Rae1β are newly identified potent stimulators of innate immunity.