Although many aspects of CD4(+)CD25(+) T regulatory (T(reg)) cell development remain largely unknown, signaling through the IL-2R represents one feature for the production of T(reg) cells. Therefore, the present study was undertaken to further define early developmental steps in the production of T(reg) cells, including a more precise view on the role of interleukin (IL)-2 in this process. After adoptive transfer of wild-type T(reg) cells into neonatal IL-2Rbeta(-/-) mice, only a small fraction of donor T(reg) cells selectively seeded the lymph node (LN). These donor T(reg) cells underwent rapid and extensive IL-2-dependent proliferation, followed by subsequent trafficking to the spleen. Thus, IL-2 is essential for T(reg) cell proliferation in neonatal LN. The number and distribution of T(reg) cells in the periphery of normal neonatal mice closely paralleled that seen for IL-2Rbeta(-/-) mice that received T(reg) cells. However, for normal neonates, blockade of IL-2 decreased T(reg) cells in both the thymus and LN. Therefore, two steps of T(reg) cell development depend upon IL-2 in neonatal mice, thymus production, and subsequent expansion in the LN.