NKG2D, together with NKp46 and NKp30, represents a major triggering receptor involved in the induction of cytotoxicity by both resting and activated human natural killer cells. In this study, we analyzed the expression and the functional relevance of MHC class I-related chain A (MICA) and UL16 binding protein (ULBP), the major cellular ligands for human NKG2D, in human tumor cell lines of different histological origin. We show that MICA and ULBP are frequently coexpressed by carcinoma cell lines, whereas MICA is expressed more frequently than ULBP by melanoma cell lines. Interestingly, the MICA(-) ULBP(+) phenotype was detected in most T cell leukemia cell lines, whereas the MICA(-) ULBP(-) phenotype characterized all acute myeloid leukemia and most B-cell lymphoma cell lines analyzed. These results, together with functional experiments, based on monoclonal antibody-mediated blocking of either NKG2D or its ligands, showed that killing of certain MICA(-) cell tumors is at least in part NKG2D dependent. Indeed, leukemic T cells as well as certain B-cell lymphomas were killed in a NKG2D-dependent fashion upon recognition of ULBP molecules. Moreover, ULBP could induce NKG2D-mediated NK cell triggering also in tumors coexpressing MICA. Our data suggest that the involvement of NKG2D in natural killer cell-mediated cytotoxicity strictly correlates with the expression and the surface density of MICA and ULBP on target cell tumors of different histotypes.