Recently several attempts to identify oxygenderived radicals in whole cells by spin trapping and electron spin resonance have been reported by using 5,5-dimethyl-1-pyrroline-N-oxide as the spin trap. In the present study, the feasibility of this method is examined. Chinese hamster V79 cells and human erythrocytes served as the test systems, while OH radicals were generated by y radiolysis. Several spin traps were used to scavenge the radicals and a distinction between exo-and endocellular ESR observable species was achieved using tri(oxalato) chromiate(I) as a line broadening agent. To distinguish between exo-and endocellular sites of radical formation, we studied the effects of high molecular weight scavengers (polyethylene glycols), which do not enter the cell. Various possible obstacles associated with trapping and detecting the radicals inside the cells were examined. The results indicate that the primary radicals react with the spin traps. However, these spin adducts decayed within the cells.Cellularly induced decay of 2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxyl radical presented the major difficulty in detecting the endogenous radicals, and potential experimental approaches to overcome this difficulty are discussed.Oxygen-derived radicals in various biological systems under physiological conditions have been identified by using spintrapping and electron spin resonance (ESR) spectrometry mainly in solutions or in in vitro systems containing subcellular components as with tissue homogenates (1)(2)(3) Chemicals. a-Phenyl-N-tert-butylnitrone (PBN), a-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a-(4-N-methylpyridinium-Ntert-butylnitrone (PyBN), and the stable nitroxide 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) were purchased from Aldrich. Polyethylene glycols (PEG) Abbreviations: PBN, a-phenyl-N-tert-butylnitrone; 4-POBN, a-(4-pyridyl-1-oxide)-N-tert-butylnitrone; DMPO, 5,5-dimethyl-1-pyrroline-N-oxide; PyBN, a-(4-N-methylpyridinium)-N-tert-butylnitrone; TEMPO, 2,2,6,6-tetramethylpiperidine-1-oxyl; Me2SO, dimethyl sulfoxide.