TCR gene therapy is adversely affected by newly formed TCRαβ heterodimers comprising exogenous and endogenous TCR chains that dilute expression of transgenic TCRαβ dimers and are potentially self-reactive. We have addressed TCR mispairing by using a modified two-chain TCR that encompasses total human CD3ζ with specificities for three different Ags. Transfer of either TCRα:CD3ζ or β:CD3ζ 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 TCRαβ:ζ does not compromise surface expression and functions of an endogenous TCRαβ. Flow cytometry fluorescence resonance energy transfer and biochemical analyses demonstrate that TCRαβ:CD3ζ is the first strategy that results in highly preferred pairing between CD3ζ-modified TCRα and β chains as well as absence of TCR mispairing between TCR:CD3ζ and nonmodified TCR chains. Intracellular assembly and surface expression of TCR:CD3ζ chains is independent of endogenous CD3γ, δ, and ε. Taken together, our data support the use of TCRαβ:CD3ζ to prevent TCR mispairing, which may provide an adequate strategy to enhance efficacy and safety of TCR gene transfer.