Mitochondrial thioredoxin reductase was purified from bovine adrenal cortex. The enzyme is a first protein component in the mitochondrial thioredoxin-dependent peroxide reductase system. The purified reductase exhibited an apparent molecular mass of 56 kDa on SDS/PAGE, whereas the native protein was about 100 kDa, suggesting a homodimeric structure. It catalysed NADPH-dependent reduction of 5, 5'dithiobis(2-nitrobenzoic acid) and thioredoxins from various origins but not glutathione, oxidized dithiothreitol, DL-alpha-lipoic acid, or insulin. Amino acid and nucleotide sequence analyses revealed that it had a presequence composed of 21 amino acids which had features characteristic of a mitochondrial targeting signal. The amino acid sequence of the mature protein was similar to that of bovine cytosolic thioredoxin reductase (57%) and of human glutathione reductase (34%) and less similar to that of Escherichia coli (19%) or yeast (17%) enzymes. Human and bovine cytosolic thioredoxin reductase were recently identified to contain selenocysteine (Sec) as one of their amino acid constituents. We also identified Sec in the C-terminal region of mitochondrial (mt)-thioredoxin reductase by means of MS and amino acid sequence analyses of the C-terminal fragment. The four-amino acid motif, Gly-Cys-Sec-Gly, which is conserved among all Sec-containing thioredoxin reductases, probably functions as the third redox centre of the enzyme, as the mitochondrial reductase was inhibited by 1-chloro-2,4-dinitrobenzene, which was reported to modify Sec and Cys covalently. It is known that mammalian thioredoxin reductase is different from bacterial or yeast enzyme in, for example, their subunit molecular masses and domain structures. These two different types of enzymes with similar activity are suggested to have evolved convergently. Our data clearly show that mitochondria, which might have originated from symbiotic prokaryotes, contain thioredoxin reductase similar to the cytosolic enzyme and different from the bacterial one.