The contribution of CD3γ to the surface expression, internalization, and intracellular trafficking of the TCR/CD3 complex (TCR) has not been completely defined. However, CD3γ is believed to be crucial for constitutive as well as for phorbol ester-induced internalization. We have explored TCR dynamics in resting and stimulated mature T lymphocytes derived from two unrelated human congenital CD3γ-deficient (γ−) individuals. In contrast to γ− mutants of the human T cell line Jurkat, which were selected for their lack of membrane TCR and are therefore constitutively surface TCR negative, these natural γ− T cells constitutively expressed surface TCR, mainly through biosynthesis of new chains other than CD3γ. However, surface (but not intracellular) TCR expression in these cells was less than wild-type cells, and normal surface expression was clearly CD3γ-dependent, as it was restored by retroviral transduction of CD3γ. The reduced surface TCR expression was likely caused by an impaired assembly or membrane transport step during recycling, whereas constitutive internalization and degradation were apparently normal. Ab binding to the mutant TCR, but not phorbol ester treatment, caused its down-modulation from the cell surface, albeit at a slower rate than in normal controls. Kinetic confocal analysis indicated that early ligand-induced endocytosis was impaired. After its complete down-modulation, TCR re-expression was also delayed. The results suggest that CD3γ contributes to, but is not absolutely required for, the regulation of TCR trafficking in resting and Ag-stimulated mature T lymphocytes. The results also indicate that TCR internalization is regulated differently in each case.