Human Vγ9Vδ2 T cells are activated through their TCR by neighboring cells producing phosphoantigens. Zoledronate (ZOL) treatment induces intracellular accumulation of the phosphoantigens isopentenyl pyrophosphate and ApppI. Few attempts have been made to use immunomanipulation of Vγ9Vδ2 lymphocytes in chronic viral infections. Although Vγ9Vδ2 T cells seem to ignore human CMV (HCMV)-infected cells, we examined whether they can sense HCMV when a TCR stimulus is provided with ZOL. Fibroblasts treated with ZOL activate Vγ9Vδ2 T cells to produce IFN-γ but not TNF. Following the same treatment, HCMV-infected fibroblasts stimulate TNF secretion and an increased production of IFN-γ, indicating that Vγ9Vδ2 cells can sense HCMV infection. Increased lymphokine production was observed with most clinical isolates and laboratory HCMV strains, HCMV-permissive astrocytoma, or dendritic cells, as well as "naive" and activated Vγ9Vδ2 cells. Quantification of intracellular isopentenyl pyrophosphate/ApppI following ZOL treatment showed that HCMV infection boosts their accumulation. This was explained by an increased capture of ZOL and by upregulation of HMG-CoA synthase and reductase transcription. Using an experimental setting where infected fibroblasts were cocultured with γδ cells in submicromolar concentrations of ZOL, we show that Vγ9Vδ2 cells suppressed substantially the release of infectious particles while preserving uninfected cells. Vγ9Vδ2 cytotoxicity was decreased by HCMV infection of targets whereas anti-IFN-γ and anti-TNF Abs significantly blocked the antiviral effect. Our experiments indicate that cytokines produced by Vγ9Vδ2 T cells have an antiviral potential in HCMV infection. This should lead to in vivo studies to explore the possible antiviral effect of immunostimulation with ZOL in this context.