Arabidopsis thaliana contains eight glutathione peroxidase (GPX) homologs (AtGPX1-8). Four mature GPX isoenzymes with different subcellular distributions, AtGPX1, -2, -5 and -6, were overexpressed in Escherichia coli and characterized. Interestingly, these recombinant proteins were able to reduce H2O2, cumene hydroperoxide, phosphatidylcholine and linoleic acid hydroperoxides using thioredoxin but not glutathione or NADPH as an electron donor. The reduction activities of the recombinant proteins with H2O2 were 2-7 times higher than those with cumene hydroperoxide. Km values for thioredoxin and H2O2 were 2.2-4.0 and 14.0-25.4 microM, respectively. These finding suggest that GPX isoenzymes may function to detoxify H2O2 and organic hydroperoxides using thioredoxin in vivo and may also be involved in regulation of the cellular redox homeostasis by maintaining the thiol/disulfide or NADPH/NADP balance.