It is clear from experimental studies in mice that T cell maturation can occur outside the thymus, especially in the intestine. There is little sound evidence so far that extrathymic T cell maturation occurs to any significant extent in human gut, and, postnatally, there is abundant evidence that the gut mucosa is an immune effector organ. Here, we describe a large population of T lymphocytes in human fetal intestinal mucosa that are proliferating (Ki67+) in the absence of foreign Ag (CD3+, Ki67+ lamina propria lymphocytes (LPL) 22±1.8% and CD3+, Ki67+ intraepithelial lymphocytes (IEL) 9.1±1.4%), that express the T cell activation markers CD103, HLA-DR, and L-selectin low, and that express mRNA transcripts for pre-TCR-α. There is also a substantial proportion of CD7+ LPLs that do not express CD3 (CD3− 7+, 14±7% of all LPLs) in the fetal gut that may be differentiating into CD3+ cells. Rearranged TCR-β transcripts of fetal LPLs, IELs, and paired blood lymphocytes were cloned and sequenced, and virtually no overlap of clonality was observed between blood and intestine, suggesting that gut T cells may not be derived from the blood. In addition, 30 days after engraftment of SCID mice with fetal intestine, CD3− 7+ cells, proliferating T cells, and pre-TCR-α transcripts were abundant, and there is a threefold increase in CD3+ IELs. These data show that in the human intestine before birth a population of precursor T cells exists that may be differentiating into mature T cells in situ