By employing electron spin resonance spectroscopy, we examined the free radicals scavenging effects of hepatic metallothionein (MT) isoforms I and II (MTs-I and II) on four types of free radicals. Solutions of 0.15 mM of MT-I and 0.3 mM of MT-II were found to scavenge the 1,1-diphenyl-2-picrylhydrazyl radicals (1.30 x 10(15) spins/ml) completely. In addition, both isoforms exhibited total scavenging action against the hydroxyl radicals (1.75 x 10(15) spins/ml) generated in a Fenton reaction. Similarly, 0.3 mM of MT-I scavenged almost 90% of the superoxide (2.22 x 10(15) spins/ml) generated by the hypoxanthine and xanthine oxidase system, while a 0.3 mM MT-II solution could only scavenge 40% of it. By using 2,2,6,6-tetramethyl-4-piperidone as a "spin-trap" for the reactive oxygen species (containing singlet oxygen, superoxide and hydroxyl radicals) generated by photosensitized oxidation of riboflavin and measuring the relative signal intensities of the resulting stable nitroxide adduct, 2,2,6,6-tetramethyl-4-piperidine-1-oxyl, we observed that MT-II (0.3 mM) could scavenge 92%, while MT-I at 0.15 mM microl/ml concentrations could completely scavenge all the reactive species (2.15 x 10(15) spins/ml) generated. The results of these studies suggest that although both isoforms of MT are able to scavenge free radicals, the MT-I appears to be a superior scavenger of superoxide and 1,1 diphenyl-2-picrylhydrazyl radicals.