Intestinal Ag exposure during neonatal life influences appropriate adult immune responses. To define the mechanisms shaping the T cell repertoire during this period, we examined T cell differentiation and receptor diversity in the intestine of human infants. Developmental phenotypes of intraepithelial and lamina propria intestinal T cells from infants aged 1 day to 2 years were assessed ex vivo by flow cytometry and in situ by triple-fluorescent immunohistochemistry. Gene recombination-specific enzymes were assessed by PCR. TCR β-chain V region gene diversity was determined by sequencing. Several different early lineage T cell populations were present neonatally: CD3+ 4− 8− T cells were present at birth and numbers decreased during the neonatal period; CD3+ 4+ 8+ T cells were present in low numbers throughout infancy; and CD3+ 4+ 8− or CD3+ 4− 8+ T cells increased with age. Very early lineage T cells, CD3− 2− 7+ and CD3− 2+ 7+, were present neonatally, but were essentially absent at 1 year. Most lamina propria T cells differentiated rapidly after birth, but maturation of intraepithelial T cells took place over 1 year. Intestinal samples from infants less than 6 mo old contained transcripts of T early α and TdT, and 15 of 19 infant samples contained mRNA for RAG-1, some coexpressing RAG-2. TCR β-chain repertoires were polyclonal in infants. Immature T cells, pre-T cells, and genes involved in T cell recombination were found in the intestine during infancy. T cell differentiation occurs within the neonatal human intestine, and the TCR repertoire of these developing immature T cells is likely to be influenced by luminal Ags. Thus, mucosal T cell responsiveness to environmental Ag is shaped in situ during early life.