Assembly of the six-chain T cell antigen receptor-CD3 complex takes place by pairwise interactions. Thus, CD3-⑀ interacts with either CD3-␥ or CD3-␦, and these dimers then associate with the TCR heterodimer (␣⅐ or ␥⅐␦) and the CD3-homodimer to constitute a full complex. We have now mapped the site in CD3-⑀ responsible for the interaction with CD3-␥ and CD3-␦ by analysis of a series of deletional mutants encompassing the most conserved regions. We found that the highly conserved juxtamembrane domain is mainly responsible for the interaction. Thus, deletion of this 16-amino acid extracellular sequence resulted in the inhibition of up to 95% of the CD3-⑀/␥ interaction. A highly conserved sequence is also present in both CD3-␥ and CD3-␦, suggesting that the domain in these two chains may reciprocally be involved in the interaction with CD3-⑀. Indeed, an immobilized synthetic peptide corresponding to the CD3-␥ sequence specifically associated to a bacterially expressed CD3-⑀ protein, suggesting the 16-amino acid domain is sufficient to promote CD3-⑀/CD3-␥ assembly. The conservation of the motif in the CD3 chains suggest that, in addition to CD3-⑀/CD3-␥ and CD3-⑀/CD3-␦ interactions, it may also mediate homotypic interactions. Indeed, it is shown that it mediates the formation of disulfide-linked homodimers and that the formation of homo-and heterodimers are mutually excluded. Finally, this domain contains a Cys-X-X-Cys sequence that resembles that of p56 lck , which is responsible for the interaction with the cytoplasmic tails of CD4 and CD8. Since the replacement of the two cysteines (Cys 97 and Cys 100 ) in CD3-⑀ by alanines strongly inhibited pair formation, the existence of a Cys-X-X-Cys motif involved in protein-protein interactions is suggested.