We have previously purified a 25-kDa thioredoxin-dependent peroxidase (TPx) from yeast and rat brain. A search for the database revealed 14 mammalian sequences, which can be divided into five distinct types I, II, III, IV, and V. This classification is supported by the distinct immunological reactivity of members of different types and by the fact that there are five distinct human sequences. We purified one representative of each of the five types of peroxiredoxins from E. coli cells containing the appropriate expression vector. Types I, II, III and IV exhibited peroxidase activity in the presence of thioredoxin. In contrast, no peroxidase activity could be demonstrated with type V protein in presence of thioredoxin. However, type V could reduce hydrogen peroxide in the presence of the nonphysiological hydrogen donor dithiothreitol, suggesting that type V is also a peroxidase. Surprisingly, glutathione could not support the peroxidase activity of type V. The distinctive property of type V is likely related to the fact that it contains only one conserved Cys and exists as a monomer even when oxidized by hydrogen peroxide while the other types exist as dimers. The catalytic efficiency of mammalian peroxiredoxins indicated by the kcat/Km is significantly lower than that of catalase or glutathione peroxidase. However, catalase is mainly in peroxisomes and glutathione peroxidase, which is mainly in cytosol, exists in low amounts in most tissues. In contrast, four out of the five mammalian peroxiredoxins are abundant (0.2-0.4 percent of total soluble protein) in cytosol. Growth factors like PDGF and EGF are known to cause a transient elevation of hydrogen peroxide. The in vivo activity of peroxiredoxins can be demonstrated by overexpressing them in NIH3T3 cells and A431 cells. Transient expression of a wild-type peroxiredoxin completely overcomes the increase in response to growth factor, whereas expression of the inactive mutant lacking the active Cys has no effect. Furthermore, overexpression of a peroxiredoxin in HeLa cells inhibits the NFKB activation elicited by hydrogen peroxide or TNFalpha. These results clearly suggest that peroxiredoxins act as a regulator of intracellular hydrogen peroxide concentration and signal transduction.