Abiotic stresses cause extensive losses to agricultural production worldwide. In this study, the effects of various abiotic stresses on the upregulation of methylglyoxal levels and glyoxalase I activities in pumpkin seedlings (Cucurbita maxima Duch.) were investigated. Most of the stresses caused significant increases in methylglyoxal level and glyoxalase I activity, white light causing the highest induction followed by salinity, chemical, drought, and heavy metal stresses. We showed that accumulation of methylglyoxal in plants under various stressful conditions is a common phenomenon, and methylglyoxal could therefore act as a signal for plants to respond to stress. The stress-induced increases in methylglyoxal level, glyoxalase I activity and Gly I transcript found in the present study suggest an important role of glyoxalase I in conferring tolerance to plants under stress conditions and showed that the glyoxalase pathway is the main detoxification pathway of methylglyoxal in plants. The multistress response of glyoxalase I gene indicates its future utility in developing tolerance to various stresses in crop plants. A cDNA encoding glyoxalase I has been isolated, subcloned and nucleotide sequence was determined. The pumpkin glyoxalase I cDNA consists of 975-bp nucleotides encoding a polypeptide of 185 amino acids having a predicted molecular weight of 20,772.14 Da. Based on the number of amino acids, it is categorized as short-type glyoxalase I and the nucleotide sequence of pumpkin glyoxalase I showed significant homology with other known glyoxalase I sequences of plants.