We report the first high-level expression of a mammalian thioltransferase (glutaredoxin) in Escherichia coli. A NcoI site (CCATGG) was introduced into the cDNA encoding pig liver thioltransferase (glutaredoxin) by site-directed mutagenesis, in which the first G of the original sequence, GCATGG, was replaced by a C. The altered cDNA was cloned into an expression vector, plasmid pKK233-2, between the unique NcoI and HindIII sites and expressed in E. coli JM105 at a high level (8% of total soluble protein) after 6 h of isopropyl-beta-D-thiogalactopyranoside induction. The soluble and unfused product was measured by the thiol-transferase thiol-disulfide exchange assay and immunoblotting analysis. The recombinant enzyme was purified to a single band as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing. The amino acid composition of the expressed enzyme agreed with that of the known sequence of pig liver thioltransferase (glutaredoxin). N-terminal sequence analysis revealed that unlike the native pig liver protein which is N-acetylated, the recombinant enzyme was unblocked at the N terminus (alanine). Various kinetic properties of the recombinant enzyme with regard to the exchange reaction were identical with those of the native enzyme.