The pre-T-cell receptor (TCR) complex, which consists of a heterodimer of the TCR beta-chain and the pre-TCR alpha-chain, is known to regulate early thymocyte development. The pre-TCR complex contains CD3 subunits as a signal-transducing molecule, but the exact subunit composition of the fully assembled pre-TCR complex remains to be elucidated. In particular, the association of the CD3 zeta-chain with the pre-TCR is controversial. In the present study, we have generated a monoclonal antibody against the cytoplasmic portion of the pre-TCR alpha-chain, and analysed a subunit composition of the pre-TCR complex. We demonstrated that the CD3 zeta-chain is physically associated with the pre-TCR in immature T cells. Thus, the result strongly supports the previous findings that CD3 zeta contributes to signalling mediated through the pre-TCR complex.