Graft-versus-host disease (GVHD) occurs in an unpredictable fashion after 30% to 50% of matched-related transplantations. The presence of increased frequencies of CD4(+)CD25(+) regulatory T cells in donor grafts has been shown to ameliorate GVHD after allogeneic transplantation in murine models. To determine whether a similar relationship exists in humans, we quantitated the coexpression of CD25 on CD4(+) and CD8(+) T cells within 60 donor grafts infused into matched siblings and examined GVHD incidence in the respective recipients. Recipients in whom GVHD developed received donor grafts containing significantly higher frequencies of CD4(+) T cells coexpressing CD25 than those who did not (median, 9.26% vs 2.22%; P =.004). Frequencies of donor graft CD8(+) T cells coexpressing CD25 were also higher (0.65% vs 0.14%; P =.002). Furthermore, transplant recipients who received grafts containing fewer CD4(+)CD25(+) and CD8(+)CD25(+) T cells were less likely to acquire acute GVHD, even though these donor-recipient pairs were similar to others with respect to relevant clinical variables. These data suggest that the coexpression of CD4 and CD25 may be insufficient to identify regulatory T cells in humans and that increased frequencies and numbers of CD25(+) T cells in donor grafts is associated with GVHD in transplant recipients.