Human γδ T cells with the TCR variable region V δ 1 occur mainly in epithelia and respond to stress-induced expression of the MHC class I-related chains A and B, which have no function in Ag presentation. MIC function as ligands for NKG2D-DAP10, an activating receptor complex that triggers NK cells, costimulates CD8 αβ and V γ 9V δ 2 γδ T cells, and is required for stimulation of V δ 1 γδ T cells. It is unresolved, however, whether triggering of V δ 1 γδ TCRs is also mediated by MIC or by unidentified cell surface components. Soluble MICA tetramers were used as a binding reagent to demonstrate specific interactions with various V δ 1 γδ TCRs expressed on transfectants of a T cell line selected for lack of NKG2D. Tetramer binding was restricted to TCRs derived from responder T cell clones classified as reactive against a broad range of MIC-expressing target cells and was abrogated when TCRs were composed of mismatched γ-and δ-chains. These results and the inability of V δ 1 γδ T cells to respond to target cells expressing the ULBP/N2DL ligands of NKG2D, which are highly divergent from MIC, indicate that MIC delivers both the TCR-dependent signal 1 and the NKG2D-dependent costimulatory signal 2. This dual function may serve to prevent erroneous γδ T cell activation by cross-reactive cell surface determinants.