Elevated expression of heme oxygenase-1 (HO-1), an intracellular enzyme that degrades heme into carbon monoxide (CO), biliverdine and free iron, has anti-inflammatory and antiapoptotic effects in diverse models. Here, we analyzed the effects of specific overexpression of HO-1 following adenovirus-mediated (AdHO-1) gene transfer in an acute cardiac allograft rejection model. The intragraft (i.g.) injection of AdHO-1 into cardiac allografts, as well as intramuscular (i.m.) or intravenous (i.v.) administration, prolonged allograft survival with, respectively, 13.3, 62.5 and 80% of the grafts surviving long term (>100 days), whereas control grafts were rejected with acute kinetics. HO-1 overexpression was associated with inhibited allogeneic responses in MLRs using graft-infiltrating leukocytes and splenocytes, but not with lymph node cells. The inhibition of splenocyte proliferation was mediated by soluble factors and was dependent on the presence of APCs, since purified T cells proliferated normally. i.v. but not i.g. AdHO-1 administration decreased the number of graft-infiltrating leukocytes, cytokine mRNA accumulation and apoptosis in transplanted hearts, whereas i.v. and i.g. AdHO-1 did not modify normal immune responses against cognate antigens, indicating that there was no general immunosuppression. These results indicate that HO-1 overexpression prolongs the survival of vascularized allografts by promoting tolerogenic mechanisms acting on allogeneic cellular immune responses.