Liver allografts in mice are accepted across MHC barriers without requirement for immunosuppressive therapy. The mechanisms underlying this phenomenon remain largely undefined. In this study, we investigated the role of Foxp3-expressing CD25+CD4+ regulatory T cells (Treg) in the induction of murine liver transplant tolerance. Foxp3+CD25 +CD4+ T cells were increased in liver grafts and recipient spleens from day 5 to day 100 posttransplantation, associated with enhanced CTLA4 and TGF-β expression and IL-4 production. Depletion of recipient CD25+CD4+ T cells using anti-CD25 mAb (250 μg/day) induced acute liver allograft rejection. This was associated with a decreased ratio of Foxp3+ Treg: T effector cells, decreased IL-4 and elevated IL-10 and IL-2 production by graft-infiltrating T cells, and reduced apoptotic activity of graft-infiltrating CD4+ and CD8+ T cells in anti-CD25-mAb-treated recipients. Thus, the data suggest that Foxp3 +CD25+CD4+Treg are involved in spontaneous acceptance of liver allografts in mice. The ratio of Treg to T effector cells appears to determine liver transplant outcome. CTLA4, IL-4, TGF-β and apoptosis of graft-infiltrating T cells are also associated with liver transplant tolerance and may contribute, at least in part, to the mechanisms of Treg-mediated immune regulation in this model.