Peripheral mechanisms preventing autoimmunity and maintaining tolerance to commensal microbiota involve CD4(+) Foxp3(+) regulatory T (T-reg) cells(1,2) generated in the thymus or extrathymically by induction of naive CD4(+) Foxp3(-) T cells. Previous studies suggested that the T-cell receptor repertoires of thymic T-reg cells and induced T-reg cells are biased towards self and non-self antigens, respectively(3-6), but their relative contribution in controlling immunopathology, such as colitis and other untoward inflammatory responses triggered by different types of antigens, remains unresolved(7). The intestine, and especially the colon, is a particularly suitable organ to study this question, given the variety of self-, microbiota- and food-derived antigens to which T-reg cells and other T-cell populations are exposed. Intestinal environments can enhance conversion to a regulatory lineage(8,9) and favour tolerogenic presentation of antigens to naive CD4(+) T cells(10,11), suggesting that intestinal homeostasis depends on microbiota-specific induced T-reg cells(12-15). Here, to identify the origin and antigen-specificity of intestinal T-reg cells, we performed single-cell and high-throughput sequencing of the T-cell receptor repertoires of CD4(+) Foxp3(+) and CD4(+) Foxp3(-) T cells, and analysed their reactivity against specific commensal species. We show that thymus-derived T-reg cells constitute most T-reg cells in all lymphoid and intestinal organs, including the colon, where their repertoire is heavily influenced by the composition of the microbiota. Our results suggest that thymic T-reg cells, and not induced T-reg cells, dominantly mediate tolerance to antigens produced by intestinal commensals.