Human γδ T cells respond to nonpeptide Ags such as pyrophosphomonoesters and alkylamines in a γδ TCR-dependent manner in the absence of other APCs. Recently, aminobisphosphonates such as pamidronate have also been shown to activate human γδ T cells. In the present study, we indicate that activation of primary γδ T cells by pamidronate strictly depends on the presence of monocyte-lineage cells, unlike that by pyrophosphomonoesters. Thus, although pamidronate induced cell clustering, proliferation, and IFN-γ production of γδ T cells in the culture of PBMC, it failed to induce any of these activities in the culture of purified primary γδ T cells. By adding back the purified monocytes, however, both cell clustering and IFN-γ production of γδ T cells by pamidronate could be restored. The pamidronate-pulsed, but not untreated, myelomonocytic line, THP-1, was capable of activating the purified γδ T cells to produce IFN-γ, which was associated with the down-regulation of γδ TCR. Furthermore, pamidronate-pulsed THP-1 cells were significantly more susceptible to γδ T cell-mediated cytotoxicity than untreated THP-1. Also, TCR-defective Jurkat T cells transfected with γδ TCR genes produced a significant level of IL-2 in response to the pamidronate-pulsed THP-1 cells. These results have suggested strongly that human γδ T cells are functionally activated via γδ TCR by aminobisphosphonate Ag presented on the surface of monocyte lineage cells rather than directly by its free form.