T lymphocyte growth is regulated by the cyclin-dependent kinase inhibitor p27 Kip1. Mice deficient in p27 Kip1 have increased proliferative responses to multiple cytokines, including IL-2, IL-4, and IL-12, but not to anti-CD3. In the absence of p27 Kip1, T cells proliferate faster than control cells, as evidenced by increased [3 H] thymidine uptake, increased cell growth and division, and an increased number of cells in S phase. Importantly, this regulation is specific for p27 Kip1 in T cells, because hyperproliferation of T cells from mice deficient in p21 Cip1/Waf1 was not observed. In vivo, there is an expansion of activated/memory CD4+ cells in p27 Kip1-deficient mice before and after immunization. Furthermore, Ag-stimulated spleen cells from immunized p27 Kip1-deficient mice demonstrated increased proliferative responses to IL-2 and increased secretion of IFN-γ. Although IL-4 stimulated proliferative responses are diminished in Stat6-deficient T cells, activated T cells from mice doubly deficient in both p27 Kip1 and Stat6 recover normal proliferative responses to IL-4. Together, these data firmly support a role for p27 Kip1 as a negative regulator of cytokine-stimulated T cell growth.