The ability of Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), a water-soluble vitamin E analogue, to prevent oxidative damages is well characterized, but the mechanisms underlying it remain unclear. The protective effect of Trolox pre-treatment on H(2)O(2)-induced toxicity might be attributed to the decreased cellular permeability to H(2)O(2) or in vitro scavenging activity of Trolox, induction of antioxidant enzymes or the direct scavenging activity of Trolox. The results obtained rule out the first and second possibilities and intracellular scavenging activity was found to be the mechanism whereby Trolox confers protection. This was confirmed by measuring protein oxidation (levels), and the observed decrease in proteasomal activity indicated that the decrease in protein carbonyls was due to Trolox scavenging activity rather than proteasome activation. In conclusion, the intracellular scavenging activity of Trolox is a key protective mechanism against H(2)O(2). These findings obtained in Schizosaccharomyces pombe, a good model organism for eukaryotic cells, can be used as standard protocols for investigating the antioxidant activity of pure or complex potential antioxidants.