We examined six Arabidopsisthaliana genes from the DJ-1/PfpI superfamily for similarity to the recently characterized bacterial and animal glyoxalases. Based on their sequence similarities, the six genes were classified into two sub-groups consisting of homologs of the human DJ-1 gene and the PH1704 gene of Pyrococcushorikoshii. Unlike the homologs from other species, all the A.thaliana genes have two tandem domains, which may have been created by gene duplication. The six AtDJ-1 proteins (a-f) were expressed in Escherichiacoli for enzymatic assays with glyoxals. The DJ-1d protein, which belongs to the PH1704 sub-group, exhibits the highest activity against methylglyoxal and glyoxal, and K-m values of 0.10 and 0.27mm were measured for these two substrates, respectively, while the corresponding k(cat) values were 1700 and 2200min(-1), respectively. The DJ-1a and DJ-1b glyoxalases exhibited higher specificity towards glyoxal. The other three proteins have either no or extremely low activity for glyoxals. For the DJ-1d enzyme, the residues, Cys120/313 and Glu19/212 at the active site and His121/314 and Glu94/287 at the oligomeric interface were mutated to alanines. As in other enzymes characterized to date, mutation of either the Cys or the Glu residues of the active site completely abolished enzyme activity, whereas mutation of the interface residues produced a variable decrease in activity. DJ-1d differs from its animal and bacterial homologs with respect to the configuration of its catalytic residues and the oligomeric property of the enzyme. When the wild-type DJ-1d enzyme was expressed in E.coli, the bacteria became resistant to glyoxals.