Abstract The T-cell subset expressing Vδ2 paired primarily with Vγ2 comprises a majority of γδ T-cells in human adult peripheral blood and expands significantly during a variety of infectious diseases. In contrast, the other subset of γδ T-cells that express Vδ1 is rare among circulating T-cells and its function is poorly understood. Here, we show that a Vγ1Vδ1 + T-cell line, 3-D, established from human peripheral blood by immortalization with Herpesvirus saimiri was able to specifically recognize tumor cells, such as K562 cells, and release cytotoxic granules containing perforin for target cell killing. Some tumor cells, including Daudi cells that are known to be susceptible to killing by Vδ2 + T-cells, were resistant to 3-D killing, implicating distinct pathways for tumor cell control by Vδ1 + and Vδ2 + T-cells. The 3-D T-cell receptor (TCR):CD3 complex reconstituted in TCR-deficient Jurkat cells was capable of transmitting signals, evidenced by activation of the interleukin 2 (IL-2) gene following ligation with anti-CD3 antibody, yet the TCR-reconstituted cells failed to produce IL-2 in response to the target cells. Thus, these results raise the possibility that some Vγ1Vδ1 + T-cells could potentially be stimulated and lyse tumor cells via ligation of TCR/CD3-unassociated molecules.