A specific subset of T cells, the FoxP3+ regulatory T cells, control effector T-cell responses to self and foreign antigens. In transplant patients, we and others have shown that high intragraft FOXP3 mRNA levels are associated with acute rejection, suggesting that immune regulation is dependent on immune activation. To study whether transplanted grafts harbor FoxP3+ T cells and to functionally analyze them, graft infiltrating lymphocytes (GILs) must be propagated from the transplanted tissue. In the present study, we analyzed whether FoxP3+ T cells can be grown from endomyocardial biopsies (EMBs; n = 5) from patients after heart transplantation during acute cellular rejection. After 18 to 21 days of culture, 0.5 to 1.0 x 10(6) GILs were cultured from the EMBs. Of these GILs, 10.6% (median; range, 1.6%-17.1%) stained positive for FoxP3. Thus Foxp3+ T cells can be grown from EMBs, providing the tools to functionally characterize these cells in depth in forthcoming studies.