Abstract The effect nitric oxide (NO ) on the stability of 5,5-dimethyl-1-pyrroline- N-oxide (DMPO) adducts has been investigated using EPR spectroscopy. We report that the DMPO/HO adduct, generated by porcine pulmonary artery endothelial cells in the presence of H 2O 2 and DMPO, or by a Fenton system (Fe(II) + H 2O 2) is degraded in the presence of the NO -donor, 2-( N, N-diethylamino)-diazenolate-2-oxide (DEANO) or by bolus addition of an aqueous solution of NO . A similar effect of DEANO was observed on other DMPO adducts, such as DMPO/ CH 3 and DMPO/ CH(CH 3)OH, generated in cell-free systems. Measurements of the loss of DMPO/HO in the presence of DEANO in aerated and oxygen-free buffers showed that in both of these settings the process obeys first-order kinetics and proceeds with similar efficacy. This indicates that direct interaction of the nitroxide with NO , rather than with NO 2 (formed from NO and O 2 in aerated media), is responsible for destruction of the spin adduct. These results suggest that the presence of NO may substantially affect the quantitative determination of DMPO adducts. We also show that NO 2 radicals, generated by a myeloperoxidase/H 2O 2/nitrite system, also degrade DMPO/HO . Because DMPO is frequently used to study generation of superoxide and hydroxyl radicals in biological systems, these observations indicate that extra caution is required when studying generation of these species in the presence of NO or NO 2 radicals.