The successful application of T cell-based immunotherapeutic applications depends on the availability of large numbers of T cells with the desired Ag specificity and phenotypic characteristics. Engineering of TCR-transferred T lymphocytes is an attractive strategy to obtain sufficient T cells with an Ag specificity of choice. However, the introduction of additional TCR chains into T cells leads to the generation of T cells with unknown specificity, due to the formation of mixed dimers between the endogenous and introduced TCR chains. The formation of such potentially autoaggressive T cells may be prevented by using gammadelta T cells as recipient cells, but the in vivo activity of such TCR-engineered gammadelta T cells has not been established. In the present study, we have investigated the in vivo functionality of TCR-transduced gammadelta T cells, in particular their Ag specific proliferative capacity, Ag specific reactivity, in vivo persistence, and their capacity to mount recall responses. The results demonstrate that alphabeta TCR engineering of gammadelta T cells forms a feasible strategy to generate Ag-specific effector T cells that do not express mixed TCR dimers. In view of increasing concerns on the potential autoimmune consequences of mixed TCR dimer formation, the testing of alphabeta TCR engineered gammadelta T cells in clinical trials seems warranted.