Electron spin resonance using spin-trapping is a useful technique for detecting direct reactive oxygen species, such as superoxide (O-2(center dot-)). However, the widely used spin trap 2,2-dimethyl-3,4-dihydro-2H-pyrrole N-oxide (DMPO) has several fundamental limitations in terms of half-life and stability. Recently, the new spin trap 2-diphenylphosphinoyl-2-methyl-3,4-dihydro-2H-pyrrole N-oxide (DPhPMPO) was developed by us. We evaluated the biological applicability of DPhPMPO to analyze O-2(center dot-) in both cell-free and cellular systems, DPhPMPO had a larger rate constant for O-2(center dot-) and formed more stable spin adducts for O-2(center dot-) than DMPO in the xanthine/xanthine oxidase (X/XO) system. In the phorbol myristate acetate-activated neutrophil system, the detection potential of DPhPMPO for O(2)(center dot-)was significantly higher than that of DMPO (k(DMPO) = 13.95 M-1 s(-1), k(DPhPMPO) = 42.4 M-1 s(-1)). These results indicated that DPhPMPO is a potentially good candidate for trapping O-2(center dot-) in a biological system. (c) 2008 Elsevier Inc. All rights reserved.