Long-lasting expansion of Vδ2^neg γδ T cells is a hallmark of cytomegalovirus (CMV) infection in kidney transplant recipients. The ligands of these cells and their role remain elusive. To better understand their immune function, we generated γδ T cell clones from several transplanted patients. Numerous patient Vδ1⁺, Vδ3⁺, and Vδ5⁺ γδ T cell clones expressing diverse Vγ chains, but not control Vγ9Vδ2⁺ T clones, displayed strong reactivity against CMV-infected cells, as shown by their production of tumor necrosis factor-α. Vδ2^neg γδ T lymphocytes could also kill CMV-infected targets and limit CMV propagation in vitro. Their anti-CMV reactivity was specific for this virus among herpesviridae and required T cell receptor engagement, but did not involve major histocompatibility complex class I molecules or NKG2D. Vδ2^neg γδ T lymphocytes expressed receptors essential for intestinal homing and were strongly activated by intestinal tumor, but not normal, epithelial cell lines. High frequencies of CMV- and tumor-specific Vδ2^neg γδ T lymphocytes were found among patients' γδ T cells. In conclusion, Vδ2^neg γδ T cells may play a role in protecting against CMV and tumors, probably through mucosal surveillance of cellular stress, and represent a population that is largely functionally distinct from Vγ9Vδ2⁺ T cells.