Members of the transforming growth factor (TGF)-beta family play pivotal roles in the control of differentiation, proliferation and tolerance in peripheral T cells. Recently, they have been implicated in thymic selection, but their role is so far not well characterized. In the present study, we demonstrate that specific thymocyte populations are under the influence of either the TGF-beta and/or Activin pathway, and transduce signals into the nucleus via phosphorylated Smad2 (pSmad2). Thymocytes in the medulla and in the subcapsular zone expressed nuclear translocated pSmad2, a hallmark of active TGF-beta/Activin receptor signaling. When analyzed at the cellular level, the pSmad2(+) cells were confined to the double-negative (DN) and single-positive (SP) subpopulations. Moreover, the most immature DN thymocytes (CD44(+)CD25(-) and CD44(+)CD25(+)) expressed higher levels of pSmad2 compared to the more mature DN. In vitro stimulation demonstrated that pure CD44(+)CD25(-), CD44(+)CD25(+) and CD44(+)CD25(+) thymocytes respond to ActivinA, while the mature CD4(+) and CD8(+) SP thymocytes respond to TGF-beta stimulation measured as enhanced phosphorylation of Smad2. Double staining of pSmad2(+) cells with either the Activin type I receptor, ALK4, or the TGF-beta type I receptor, ALK5, demonstrated that pSmad2(+) DN cells exhibited high levels of immunoreactivity to ALK4 and moderate levels of immunoreactivity to the TGF-beta-responsive ALK5 receptor. In sharp contrast, the SP pSmad2(+) cells were predominately ALK5(+). Collectively, our results demonstrate that early and late thymocytes express pSmad2 in the nuclei in vivo. The functional experiments in vitro suggest that members of the TGF-beta family (TGF-beta or Activin) may play important non-redundant roles during different stages of thymopoiesis.