Human Vγ9Vδ2 γδ T cells are selectively activated by bacterial phosphoantigens and aminobisphosphonates and exert potent cytotoxicity toward various tumor cells. In this study we have characterized the cytotoxic reactivity of γδ T cell lines established from healthy donors by stimulation with aminobisphosphonate alendronate toward melanoma MeWo and pancreatic adenocarcinomas Colo357 and PancTu1 lines in vitro and in vivo upon adoptive transfer into SCID mice. Lysis of all tumor cells was enhanced when γδ effector cells were preactivated with phosphoantigens. Recognition of MeWo was TCR dependent, as shown by anti-TCR Ab blockade, whereas only the phosphoantigen-mediated increased, but not the basal, lysis of Colo357 and PancTu1 was inhibited by anti-TCR Ab. Furthermore, lysis of Colo357, but not that of MeWo or PancTu1, was completely inhibited by the pan-caspase inhibitor zVAD, indicating different recognition and effector mechanisms involved in the γδ T cell/tumor cell interactions. Upon transfer into SCID mice, alendronate-activated γδ T cells given together with IL-2 and alendronate significantly prolonged the survival of SCID mice inoculated with human tumor cells. The best results were thus obtained when γδ T cells were repetitively given five times over a period of 30 days. With this protocol, human γδ T cells prolonged the mean survival of mice inoculated with MeWo melanoma from 28.5 to 87.3 days (p< 0.0001) and in the case of PancTu1 adenocarcinoma from 23.0 to 48.4 days (p< 0.0001). We conclude that an effective γδ T cell-based immunotherapy might require activation of endogenous γδ T cells with aminobisphosphonate (or phosphoantigen) and IL-2, followed by adoptive transfer of in vitro expanded γδ T cells.