CD3epsilongamma and CD3epsilondelta are noncovalent heterodimers; each consists of Ig-like extracellular domains associated side-to-side via paired terminal beta-strands that are linked to individual subunit membrane proximal stalk segments. CD3epsilon, CD3gamma, and CD3delta stalks contain the RxCxxCxE motif. To investigate the functional importance of a CD3 stalk and terminal beta-strand, we created a CD3gamma double mutant CD3gamma(C82S/C85S) and a CD3gamma beta-strand triple mutant CD3gamma(Q76S/Y78A/Y79A) for use in retroviral transduction of lymphoid progenitors for comparison with CD3gammawt. Although both mutant CD3gamma molecules reduced association with CD3epsilon in CD3epsilongamma heterodimers, CD3gamma(Q76S/Y78A/Y79A) abrogated surface TCR expression whereas CD3gamma(C82S/C85S) did not. Furthermore, CD3gamma(C82S/C85S) rescued thymic development in CD3gamma(-/-) fetal thymic organ culture. However, the numbers of double-positive and single-positive thymocytes after CD3gamma(C82S/C85S) transduction were significantly reduced despite surface pre-TCR and TCR expression comparable to that of CD3gamma(-/-) thymocytes transduced in fetal thymic organ culture with a retrovirus harboring CD3gammawt cDNA. Furthermore, double-negative thymocyte development was perturbed with attenuated double-negative 3/double-negative 4 maturation and altered surface-expressed CD3epsilongamma, as evidenced by the loss of reactivity with CD3gamma N terminus-specific antisera. Single histidine substitution of either CD3gamma stalk cysteine failed to restore CD3epsilongamma association and conformation in transient COS-7 cell transfection studies. Thus, CD3gamma(C82) and CD3gamma(C85) residues likely are either reduced or form a tight intrachain disulfide loop rather than contribute to a metal coordination site in conjunction with CD3epsilon(C80) and CD3epsilon(C83). The implications of these results for CD3epsilongamma and TCR structure and signaling function are discussed.