Methylglyoxal (MG), a cytotoxic by-product produced mainly from triose phosphates, is used as a substrate by glyoxalase I. In this paper, we report on the estimation of MG level in plants which has not been reported earlier. We show that MG concentration varies in the range of 30-75 microM in various plant species and it increases 2- to 6-fold in response to salinity, drought, and cold stress conditions. Transgenic tobacco underexpressing glyoxalase I showed enhanced accumulation of MG which resulted in the inhibition of seed germination. In the glyoxalase I overexpressing transgenic tobacco, MG levels did not increase in response to stress compared to the untransformed plants, however, with the addition of exogenous GSH there was a decrease in MG levels in both untransformed and transgenic plants. The exogenous application of GSH reduced MG levels in WT to 50% whereas in the transgenic plants a 5-fold decrease was observed. These studies demonstrate an important role of glyoxalase I along with GSH concentration in maintaining MG levels in plants under normal and abiotic stress conditions.