The substrate specificity of thioltransferase (thiol:disulfide oxidoreductase) purified from rat liver cytosol (Axelsson, K., Eriksson, S. and Mannervik, B. (1978) Biochemistry 17, 2978-2984) has been investigated. Various low-molecular-weight thiols were found to be substrates in the scission of sulfur-sulfur bonds in cystamine and glutathione disulfide as well as in mixed disulfides of proteins and glutathione. Protein sulfhydryl groups could also serve as thiol substrates in enzyme-catalyzed thiol-disulfide interchange. Thiol-transferase also catalyzed the reduction of sulfur-sulfur bonds in disulfide-containing proteins by glutathione provided that the proteins had been denatured by proteolysis or urea treatment. It is proposed that thioltransferase may have a role in the intracellular protein degradation by cleavage of disulfide bonds. The finding that thioltransferase catalyzes the reversible formation of mixed disulfides of glutathione and the bulk of proteins in rat liver cytosol is relevant for the catalysis of changes in the 'glutathione status' in the cell. In conclusion, the results of the present investigation show that cytoplasmic thiol-transferase has a very broad substrate specificity, which implies that the enzyme may participate in a multitude of thiol-disulfide interchange reactions.