Heme oxygenase 1 (HO-1) is known to inhibit apoptosis by regulating cellular prooxidant iron. This study demonstrates an additional mechanism by which HO-1 inhibits apoptosis: the generation of carbon monoxide (CO). Overexpression of HO-1 or induction of HO-1 expression by heme protects endothelial cells (ECs) from apoptosis. When HO-1 enzymatic activity is blocked or the action of CO is inhibited, HO-1 no longer prevents EC apoptosis. Exogenous CO, when used in the absence of HO-1 activity, substitutes for HO-1 in preventing EC apoptosis. The mechanism of action of HO-1/CO is dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Expression of HO-1 or exposure of ECs to exogenous CO enhances p38 MAPK activation by TNF-α. Specific inhibition of p38 MAPK activation abrogates the antiapoptotic effect of HO-1. These findings show that the antiapoptotic effect of HO-1 in ECs is mediated by CO and specifically via the activation of p38 MAPK by CO.Heme oxygenase 1 (HO-1) is known to inhibit apoptosis by regulating cellular prooxidant iron. This study demonstrates an additional mechanism by which HO-1 inhibits apoptosis: the generation of carbon monoxide (CO). Overexpression of HO-1 or induction of HO-1 expression by heme protects endothelial cells (ECs) from apoptosis. When HO-1 enzymatic activity is blocked or the action of CO is inhibited, HO-1 no longer prevents EC apoptosis. Exogenous CO, when used in the absence of HO-1 activity, substitutes for HO-1 in preventing EC apoptosis. The mechanism of action of HO-1/CO is dependent on the activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Expression of HO-1 or exposure of ECs to exogenous CO enhances p38 MAPK activation by TNF-α. Specific inhibition of p38 MAPK activation abrogates the antiapoptotic effect of HO-1. These findings show that the antiapoptotic effect of HO-1 in ECs is mediated by CO and specifically via the activation of p38 MAPK by CO.