The human T-cell receptor-CD3 complex consists of at least eight polypeptide chains; CD3 gamma epsilon- and delta epsilon-dimers associate with the disulfide linked alpha beta- and zeta zeta-dimers to form a functional receptor complex. The exact structure of this complex is still unknown. We now have examined the interaction between CD3 gamma and CD3 epsilon in human T-cells. For this purpose, we have generated site-directed mutants of CD3 gamma that were introduced in human T-cells defective in CD3 gamma expression. Cell-surface and intracellular expression of the introduced CD3 gamma chains was determined, as was the association with CD3 delta, CD3 epsilon, and the T-cell receptor. Although the introduction of wild type CD3 gamma and CD3 gamma (78Y-F) fully restored T-cell receptor assembly and expression, the introduction of CD3 gamma (82C-S), CD3 gamma (85C-S), and CD3 gamma (76Q-E) all resulted in an impaired association between CD3 gamma and CD3 epsilon and a lack of cell-surface expressed CD3 gamma. Finally, the introduction of CD3 gamma (76Q-L) and CD3 gamma (78Y-A) restored the expression of TCR-CD3 delta epsilon gamma epsilon zeta(2) complexes, although the association between CD3 gamma and CD3 epsilon was impaired. These results indicate that several amino acids in CD3 gamma are essential for an optimal association between CD3 gamma and CD3 epsilon and the assembly of a cell-surface expressed TCR-CD3 delta epsilon gamma epsilon zeta(2) complex.