Mutant αβ TCRs were generated by replacing domains of the α and β chain constant regions with homologous domains from TCR δ and γ chains, respectively. Chimeric TCRs in which the α chain contains TCR δ chain sequences within the connecting peptide domain are unresponsive to alloantigens and superantigens, and have defective interactions with the CD3/ζ complex. Although these antigen-unresponsive TCRs undergo ζ chain phosphorylation upon stimulation with superantigen, they do not generate a full signal capable of producing IL-2. Mutant TCRs acquire signaling activity with a combination of superantigen and calcium ionophore, indicating a defect in calcium-mediated signaling. Finally, a conserved motif, FETDxNLN, present in the α chain connecting peptide domain, is disrupted in all signaling-defective TCRs. This conserved α chain connecting peptide motif might mediate the transfer of signals from the αβ heterodimer to the CD3/ζ complex.