We examined the appearance of intestinal intraepithelial lymphocytes (IEL) during the first 12 wk of life to gain insight into postnatal factors that contribute to the differences found between IEL in the large and small intestines of adult mice. Intestinal T cells were very infrequent at birth, but increased in number in the large and small intestine during the first 4 wk of life and then stabilized. The small intestinal epithelium at 2 wk of age contained mostly T cell receptor (TCR) alphabeta+, CD2+ T cells, unlike IEL in adult mice, which were composed of nearly equal proportions of CD2-, TCR alphabeta+ and TCR gammadelta+ cells. Between 2 and 3 wk of age, TCR gammadelta+, CD2- IEL increased greatly in the small intestine, whereas TCR alphabeta+ cells expressing CD2 decreased. By contrast, IEL in the large intestine at 2 and 3 wk of age were mostly TCR alphabeta+, CD2+ T cells similar to large intestinal IEL in adult mice. And finally, the expression of CD69 increased earlier and to higher levels on TCR alphabeta+ and TCR gammadelta+ IEL in the small intestine than in the large intestine. Our results demonstrate that IEL in the large and small intestine are phenotypically similar during suckling and that differences between these populations are established after weaning. Furthermore, the earlier accumulation of IEL with an activated adult IEL phenotype in the small intestine suggests that these T cells mature or expand in the gut and contribute to the maturation of immune function during postnatal life in mice.