In 2 consecutive prospective clinical trials, we evaluated the efficacy of sirolimus together with a calcineurin inhibitor (cyclosporine or tacrolimus) and low-dose methotrexate for prevention of graft-versus-host disease (GVHD) after unrelated hematopoietic cell transplantation (HCT). Nine patients received sirolimus with cyclosporine, and 17 received sirolimus with tacrolimus. The incidence of grade II-IV GVHD was 77%, with the median onset at day 7 after HCT. Because of toxicity, administration of sirolimus was discontinued earlier than planned in 11 patients, but after the onset of GVHD. Three patients developed renal failure requiring hemodialysis. Accrual in both studies was terminated because of lack of efficacy. In these studies, the addition of sirolimus to regimens containing a calcineurin inhibitor and methotrexate appeared to cause toxicity and provided no detectable improvement in preventing GVHD.