Prostaglandin H synthase (PHS) is a dual enzyme with cyclo-oxygenase and peroxidase activities. The nitric oxide (NO) donors, sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and spermine NONOate (SPER/NO), activated both cyclo-oxygenase and peroxidase activities of PHS. SNP activated PHS by increasing Vmax without affecting Km, the activation constants being 1.0 mM for cyclo-oxygenase and 1.3 mM for peroxidase. Analysis of progress curves and absorption spectra of PHS suggested that NO released from SNP interacted with the heme at the active site of the enzyme. Moreover, SNP counteracted the peroxide-induced inactivation of PHS, suggesting that the interplay between the intracellular peroxide and NO is critical in tuning PHS activity in cells.