The TCR/CD3 complex is composed of six subunits which are expressed on the cell surface in a coordinate fashion after assembly in the endoplasmic reticulum (ER). The TCR/CD3 complex is assembled after a series of pairwise interactions involving the formation of dimers of CD3 epsilon with either CD3 gamma or CD3 delta. These dimers assemble with TCR alpha and TCR beta chains, and finally, the CD3 zeta homodimer is added to allow export of the full complex from the ER. A model has been proposed suggesting that during assembly the CD3 epsilon/CD3 gamma dimer interacts exclusively with TCR beta and the CD3 epsilon/CD3 delta dimer with TCR alpha to form a complex with a single TCR alpha/beta heterodimer. We show in this study, by immunoprecipitation and two-dimensional gel electrophoresis, that in the human T cell line Jurkat as well as in total human thymocytes, this preferential interaction does not occur and instead, the CD3 epsilon/CD3 gamma and CD3 epsilon/CD3 delta dimers associate with both TCR chains simultaneously and indistinctly. These data are confirmed by the analysis of the TCR alpha-negative T cell line MOLT-4 in which TCR beta is found separately associated with CD3 epsilon/CD3 gamma and with CD3 epsilon/CD3 delta dimers. Indirectly, our results support a model of stoichiometry in which two TCR alpha/beta heterodimers are present in a TCR/CD3 complex. Furthermore, immunoprecipitation with anti-CD3 gamma and anti-CD3 delta antibodies from 1% NP40 and 1% Brij96 cell lysates showed that these subunits form independent partial complexes which are cross-linked through the CD3 zeta homodimer. This suggests that CD3 zeta mediates the interaction between both TCR alpha/beta heterodimers contained in the double TCR complex. Further proof for this hypothesis is obtained after analysis of a Jurkat cell transfectant containing a point mutation in the transmembrane domain of TCR beta that impairs the association of CD3 zeta. In this mutant cell line, unlike a control line with wild-type TCR beta, the CD3 gamma- and CD3 delta-containing complexes were found completely independent. Altogether, these results support a model of TCR/CD3 assembly and stoichiometry in which two TCR-alpha/beta heterodimers form two hemicomplexes containing either CD3 epsilon/gamma or CD3 epsilon/delta dimers which become associated via the CD3 zeta homodimer.