Small intestinal cryptopatches (CP) are the major anatomic site for extrathymic differentiation by precursors destined to become intestinal intraepithelial T lymphocytes (IEL). We found that mice deficient in CCR6 exhibited a 2.7-fold increase in the number of alphabeta TCR IEL, but little or no expansion of gammadelta TCR IEL. Among the alphabeta TCR IEL subsets, the CD4- CD8alphaalpha+ and CD4+ CD8alphaalpha+ subsets were preferentially expanded in CCR6 null mice. Because some CD8alphaalpha+ IEL can arise through extrathymic differentiation in CP, we investigated CCR6 expression by T lymphocyte precursors undergoing extrathymic differentiation in intestinal CP. In sections of CP, 50-60% of c-kit+ precursors were CCR6+. CD11c(+) cells concentrated at the periphery of CP did not express CCR6. A subset of c-kit+, Lin- cells in lamina propria suspensions was CCR6+, but CCR6 was absent from c-kit+ precursors in bone marrow. CCR6 was absent from the vast majority of mature IEL. CCR6 is present on lymphocyte precursors in cryptopatches, expressed transiently during extrathymic IEL development, and is required for homeostatic regulation of intestinal IEL.