We have examined the ontogeny of mouse intestinal intraepithelial lymphocytes (IEL) to gain insight into the possible extrathymic differentiation of T cells in the intestine and to identify intermediate stages of this pathway. Intestinal T cells are undetectable or very infrequent at birth, with a dramatic increase in cell number within a few weeks. The small intestinal epithelium at 2 weeks of age contains mostly TCR-cells and some TCR αβ+ cells, a large majority of which are CD4 and CD8 negative. Between 2 and 3 weeks, several subpopulations increase, particularly TCR γδ+, CD8αα+ cells. Therefore, unlike T cell ontogeny in the thymus, γδ+ cells do not appear in the small intestine prior to TCR αβ+ IEL. There is no accumulation of TCRαβ+, CD4, CD8αα double positive cells or CD8αα single positive cells early in ontogeny, suggesting that cells with this phenotype are not precursors of CD4 or CD8 single positive IEL. The presence of so called cryptopatches, CD3-, CD117+ (c-kit+) cell aggregates in the lamina propria of the small intestine, prior to and coincident with the appearance of TCR+ IEL, suggests that some intestinal IEL may arise in this site. By contrast, large intestinal IEL are comprised of adult proportions of TCR αβ+ and γδ+ cells as early as 2 weeks postnatal age. Our studies show that populations of IEL are differentially established in the intestine during neonatal development with a pivotal point at about the time of weaning. It is likely that some IEL mature via an extrathymic pathway and that this pathway is centered in the intestine. Robert Wood Johnson Foundation (VC) and PO1 DK46763 (MK).