To evaluate potential catalytic mechanism for thioltransferase thiol-disulfide exchange reactions, seven pig liver mutants were constructed by site-directed mutagenesis. All the expressed enzymes, including wild-type and mutants with the exception of the inactive mutant, ETT-C22S, were variably inhibited by iodoacetamide, and similar results were obtained when these enzymes were preincubated with GSH. However, when preincubated with S-sulfocysteine or hydroxyethyl disulfide, the activity of the enzymes was totally or partially protected against inhibition by iodoacetamide, with the exception of the mutants, ETT-C25S and ETT-C25A. When simultaneously pretreated with GSH and S-sulfocysteine, all enzymes were highly protected. Isoelectric focusing analysis of the above preincubation mixtures showed that different enzyme-substrate intermediates occurred. Using radioactively labeled substrates, [U-14C]cystine and [glycine-2-3H] GSH, enzyme-substrate intermediates were detected. These data indicate that reduced thioltransferase reacts first with disulfide substrates, then with a thiol substrate, e.g. GSH. The formation of either enzyme-substrate mixed disulfide or protein intramolecular disulfide protected the enzyme from inactivation by iodoacetamide. Based on the experimental results, alternative methods of the catalytic mechanism for thioltransferases are proposed.