The present study investigates the involvement of nitric oxide (NO) and cyclic GMP (cGMP) in antioxidant cellular protection induced by aspirin. A 24-h incubation with hydrogen peroxide markedly reduced viability of cultured endothelial cells. Preincubation with aspirin (3-30 µmol/L) protected endothelial cells from hydrogen peroxide-mediated toxicity and increased viability in a concentration-dependent fashion by up to 95% of control. This effect was specific in that other nonsteroidal anti-inflammatory drugs, such as salicylate or indomethacin, did not alter hydrogen peroxide toxicity. Aspirin-induced endothelial protection was abrogated in the presence of the NO scavenger PTIO (30 µmol/L) and the inhibitor of soluble guanylyl cyclase ODQ (1 µM). Moreover, the L-arginine antagonist L-NMMA (25 µmol/L), but not its D-enantiomer, led to complete inhibition of aspirin-dependent cytoprotection. In agreement with this, aspirin enhanced NO synthase activity (citrulline formation) and intracellular cGMP accumulation in endothelial cells. Protein expression of endothelial NO synthase remained unaffected in the presence of aspirin. Aspirin had no effect on cGMP levels in RFL-6 cells that are devoid of NO synthase activity but rich in soluble guanylyl cyclase. Moreover, cGMP increases by both aspirin or exogenous nitric oxide were synergistically enhanced in the presence of YC-1, an NO-independent activator of soluble guanylyl cyclase. These observations corroborate the involvement of nitric oxide in aspirin-dependent cGMP stimulation. Our data demonstrate that endothelial NO synthase is a site of action of aspirin and that the NO/cGMP system assumes a crucial function in mediating the cytoprotective action of aspirin.