PhenylN-tert-butyl nitrone (PBN) is commonly employed in spin-trapping studies. We report here evidence that PBN in aqueous solutions is decomposed by two pathways leading to the generation of nitric oxide ('NO). The first pathway is by hydrolysis of PBN, which is strongly catalyzed by ferric iron. The second pathway is via PBN-hydroxyl radical adduct formation. NO was trapped in the presence of cysteine and ferrous iron to form a [(cys)2Fe(NO)2]−3complex, which was measured by use of electron paramagnetic resonance (EPR) spectroscopy. A concomitant metabolite, benzaldehyde, was detected from both reaction mixtures. We propose that PBN is hydrolyzed by Fe3+or attacked by hydroxyl radical, leading eventually to a common transient species,tert-butyl hydronitroxide [t-BuN(O')H], which is further oxidized to a 'NO source,t-BuNO. Our data imply that PBN may decompose to 'NO when used in biological models with oxidative stress conditions.