Previous reports have suggested that protein disulfide isomerases (PDIs) have transglutaminase (TGase) activity [1]. The structural basis of this reaction has not been revealed. We demonstrate here that Caenorhabditis elegans PDI-3 can function as a Ca2+-dependent TGase in assays based on modification of protein- and peptide-bound glutamine residues. By site-directed mutagenesis the second cysteine residue of the -CysGlyHisCys- motif in the thioredoxin domain of the enzyme protein was found to be the active site of the transamidation reaction and chemical modification of histidine in their motif blocked TGase activity. (C) 2003 Elsevier Science (USA). All rights reserved.