Naturally arising CD4⁺CD25⁺ regulatory T cells play a pivotal role in the prevention of autoimmunity and in the induction of donor-specific transplantation tolerance. Harnessing regulatory cells for potential adoptive cell therapy is hampered by their lack of antigen-specificity and their limited numbers. Here we describe the generation and expansion of murine CD4⁺CD25⁺ T cells with antigen-specificity for an K^d peptide as potential reagents for adoptive cell therapy in promoting donor-specific transplantation tolerance. Using bone marrow-derived autologous dendritic cells pulsed with the K^d peptide, we generated T cell lines from purified CD4⁺CD25⁺ T cells from C56BL/6 mice. The T cell lines expressed high level of CD25 and low level of CD45RB and CD69. They maintained the expression of CD62L, GITR, CTLA-4 and more importantly FoxP3. The CD4⁺CD25⁺ T cell lines were anergic after TCR stimulation and produced little cytokine such as IL-2 and IFN-γ. Importantly, they were more potent than freshly isolated CD4⁺CD25⁺ T cells in suppressing proliferation and cytokine secretion by effector CD4⁺ T cells. Furthermore, the CD4⁺CD25⁺ T cell lines could be expanded to large cell numbers and maintained in culture up to 1 year. The K^d-specific CD4⁺CD25⁺ T cell lines will be invaluable in devising a strategy for the induction of cardiac transplantation tolerance in wild-type B6 mice carrying a full mismatch BALB/c heart.