Nitric oxide-releasing nonsteroidal anti-inflammatory drugs (NO-NSAIDs) donate nitric oxide in biological systems through as yet unidentified mechanisms. cGMP, a marker of intracellularly generated NO, was increased by NO-naproxen in LLC-PK-1 cells. The increase in cGMP is fully attributable to the release of NO because it is diminished by incubation of cells with the NO scavenger hemoglobin and enhanced by superoxide dismutase, which is known to stabilize the short-lived nitric oxide free radical. Pretreatment with either glyceryl trinitrate (GTN) or NO-naproxen attenutes the cGMP response to both agents, which demonstrates that NO-NSAIDs are bioactivated through pathways that are also responsible for NO formation from nitric acid esters such as GTN. Incubation of human endothelial cells with different NO-NSAIDs led to increased expression of heme oxygenase(HO)-1 mRNA and protein. In contrast, the NO-free parent compounds and rofecoxib, an inhibitor of COX-2 left HO-1 expression unaltered. Pretreating endothelial cells with NO-NSAIDs at concentrations that were also effective in HO-1 induction reduced NADPH-dependent production of oxygen radicals. Antioxidant activity in endothelial cells persisted after NO-NSAIDs have been removed from the incubation medium. The HO-1 metabolite bilirubin, when added exogenously to the cells at low micromolar concentrations virtually abolished NADPH-dependent oxidative stress. NO-NSAID-induced blockade of free radical formation was rescued in the presence of the HO inhibitor, SnPP. Similar results were obtained in human gastric mucosal cells. The results demonstrate that HO-1 is a novel target of NO-NSAIDs and mediates antioxidant actions of these compounds in vascular and gastric tissue.