Plants contain several genes encoding thioredoxins (Trxs), small proteins involved in the regulation of the activity of manyenzymes through dithiol-disulfide exchange. In addition to chloroplastic and cytoplasmic Trx systems, plant mitochondriacontain a reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase and a specific Trx o, and to date,there have been no reports of a gene encoding a plant nuclear Trx. We report here the presence in pea (Pisum sativum)mitochondria and nuclei of a Trx isoform (PsTrxo1) that seems to belong to the Trx o group, although it differs from this Trxtype by its absence of introns in the genomic sequence. Western-blot analysis with isolated mitochondria and nuclei,immunogold labeling, and green fluorescent protein fusion constructs all indicated that PsTrxo1 is present in both cellcompartments. Moreover, the identification by tandem mass spectrometry of the native mitochondrial Trx after gel filtrationusing the fast-protein liquid chromatography system of highly purified mitochondria and the in vitro uptake assay intoisolated mitochondria also corroborated a mitochondrial location for this protein. The recombinant PsTrxo1 protein has beenshown to be reduced more effectively by the Saccharomyces cerevisiae mitochondrial Trx reductase Trr2 than by the wheat(Triticum aestivum) cytoplasmic reduced nicotinamide adenine dinucleotide phosphate-dependent Trx reductase. PsTrxo1 wasable to activate alternative oxidase, and it was shown to interact with a number of mitochondrial proteins, includingperoxiredoxin and enzymes mainly involved in the photorespiratory process.