The existence of E. coli extended thioredoxin, which contains 19 amino acid residues longer than the well-known thioredoxin of 108 amino acid residues, has previously been confirmed by site-directed mutagenesis. An initiation mutant of thioredoxin gene (trxAE) gives the production of a mutant extended thioredoxin, because the second ATG codon (for methionine) was converted to CTG (for leucine). In order to purify the mutant extended thioredoxin, the protein was labeled with ^35S-methionine using T7 RNA polymerase/promoter system, and traced by radioactivity through purification steps such as ammonium sulfate fractionation, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. The purified mutant extended thioredoxin was characterized by the affinity for E. coli thioredoxin reductase, insulin reduction, and heat stability.