We investigated the hypoxanthine (HPX)-xanthine oxidase (XOD) reaction by examining the chemiluminescence (CL) response mediated by a luminol analog, 8-amino-5-chloro-7-phenylpyrido[3,4-djpyridazine-1,4-(2H,3H)-dione sodium salt (L-012). It was found that addition of a high concentration of dimethyl sulfoxide (DMSO), a potent OH scavenger, could not completely reduce the CL response. This result suggests the existence of an unknown reactive oxygen intermediate other than O-2(-center dot) and (OH)-O-center dot. We further examined the HPX-XOD reaction and the nicotinamide adenine dinucleotide phosphate (NADPH) oxidation reaction by applying an electron spin resonance (ESR) spin-trapping method. In both reaction systems, similar responses were observed. That is, addition of DMSO increased the formation of 5.5-dimethyl-1-pyrroline N-oxide (DMPO) -OOH in a concentration-dependent manner. This indicates that scavenging of (OH)-O-center dot increases the detected O-2(-center dot) level, further suggesting the existence of an intermediate oxygen species derived from O-2(-center dot) and (OH)-O-center dot. One candidate for this species is HOOOH, presumably formed in the following way. O-2(center dot) + H+ + (OH)-O-center dot -> HOOOH