Cereal proteins are known to cause allergic reactions such as Baker's asthma and severe atopic dermatitis to certain populations. In rice allergy, proteins with molecular masses of 14-16, 26, 33, and 56 kDa have been demonstrated to be potentially allergenic. In this study, to identify and characterize the 33-kDa allergen, designated Glb33, this protein was first purified to homogeneity, and its cDNA clone was isolated. When expressed in Escherichia coli, the recombinant Glb33 was shown to be as reactive as the native Glb33 with mouse IgG and patients' IgE antibodies to Glb33. The Glb33 cDNA coded for a protein of 291 amino acids with two 120-amino acid residue repeats, and the amino acid sequence showed similarity to glyoxalase I from various organisms, including human, plant, yeast, and bacterium. As expected, both native Glb33 purified from rice seeds and the recombinant protein had glyoxalase I activity that catalyzes condensation of methylglyoxal and glutathione into S-lactoylglutathione. However, Glb33 had a higher sequence identity to the bacterial glyoxalase I rather than to known plant and yeast enzymes. Both the Glb33 transcript and the protein were detected not only in maturing seeds of rice but also in its stem and leaf. Taken all together, the rice allergen, Glb33, was identified to be a novel type of plant glyoxalase I that is expressed in various plant tissues, including maturing seeds.