To assess whether regulatory T cells are present in rejecting human cardiac allografts, we performed functional analyses of graft lymphocytes (GLs) expanded from endomyocardial biopsies (EMB; n = 5) with histological signs of acute cellular rejection. The GL cultures were tested for their proliferative capacity and regulatory activity on allogeneic-stimulated peripheral blood mononuclear cells (PBMC) of the patient (ratio PBMC:GLs = 5:1). Three of these GL cultures were hyporesponsive to donor antigens and suppressed the antidonor proliferative T-cell response of PBMC, but not the anti-third-party response. Interestingly, it was the CD8(+) GL subset of these cultures that inhibited the antidonor response (65-91% inhibition of the proportion of proliferating cells); the CD4(+) GLs of the expanded GL cultures were not suppressive. In conclusion, CD8(+) GLs expanded from rejecting human cardiac allografts can exhibit donor-specific immune regulatory activities in vitro. We suggest that during acute cellular rejection, GLs may not only consist of graft-destructing effector T cells, but also of cells of the CD8(+) type with the potential to specifically inhibit antidonor immune reactivity.