The protease dipeptidylpeptidase IV (CD26) provides an alternative activation pathway for T lymphocytes and is involved in several aspects of T cell function. Activation via CD26 requires the expression of the T cell receptor (TcR)/CD3 complex. Here we have investigated the role of the TcR zeta chain for T cell activation via CD26. T cell hybridomas expressing TcR with various deletions in the CD3 zeta chain were transfected with a CD26 cDNA and the response of the transfected cells to anti-CD26 monoclonal antibodies was tested. Our data show that the zeta chain is essential and that at least one YXXL motif in the cytoplasmic tail of the zeta chain is required for CD26-mediated signaling. Other TcR components do not replace the zeta chain.