2-2'-Pyridylisatogen (PIT) has been reported to be a relatively selective irreversible antagonist of responses to adenosine 5'-triphosphate (ATP) in some smooth muscle preparations and to be an allosteric modulator of responses to ATP at recombinant P2Y receptors from chick brain. PIT is also a potent inhibitor of mitochondrial oxidative phosphorylation. However, the compound has a unique nitrone structure, so PIT was compared with dimethyl-pyrroline-N-oxide (DMPO) as a spin trapping agent for superoxide and hydroxyl radicals using electron spin resonance (ESR). PIT was found to be a potent spin trapper of both hydroxyl and superoxide radicals. PIT was more potent than DMPO to trap the hydroxyl radical forming an adduct which was more stable than the DMPO adduct in aqueous media. PIT was an effective spin trap of hydroxyl radical in aqueous buffer at pH 7.4. PIT more slowly trapped the superoxide anion but at concentrations where DMPO trapped none.