TCR gene therapy is adversely affected by newly formed TCRalphabeta heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCRalphabeta dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3zeta with specificities for three different Ags. Transfer of either TCRalpha:CD3zeta or beta:CD3zeta genes alone does not result in surface expression, whereas transfer of both modified TCR chains results in high surface expression, binding of peptide-MHC complexes and Ag-specific T cell functions. Genetic introduction of TCRalphabeta:zeta does not compromise surface expression and functions of an endogenous TCRalphabeta. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCRalphabeta:CD3zeta is the first strategy that results in highly preferred pairing between CD3zeta-modified TCRalpha and beta chains as well as absence of TCR mispairing between TCR:CD3zeta and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3zeta chains is independent of endogenous CD3gamma, delta, and epsilon. Taken together, our data support the use of TCRalphabeta:CD3zeta to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.