Hydrogen sulfide (H(2)S) is a gasotransmitter with multifaceted physiological functions, including the regulation of glucose metabolism. Methylglyoxal (MG) is an intermediate of glucose metabolism and plays an important role in the pathogenesis of insulin resistance syndromes. In the present study, we investigated the effect of MG on H(2)S synthesis and the interaction between these two endogenous substances. In cultured vascular smooth muscle cells (VSMCs), MG (10, 30, and 50 microM) significantly decreased cellular H(2)S levels in a concentration-dependent manner, while H(2)S donor, NaHS (30, 60, and 90 microM), significantly decreased cellular MG levels. The expression level and activity of H(2)S-producing enzyme, cystathionine gamma-lyase (CSE), were significantly decreased by MG treatment. NaHS (30-90 microM) significantly inhibited MG (10 or 30 microM)-induced ROS production. Cellular levels of GSH, cysteine, and homocysteine were also increased by MG or NaHS treatment. Furthermore, direct reaction of H(2)S with MG in both concentration- and time-dependent manners were observed in in vitro incubations. In conclusion, MG regulates H(2)S level in VSMCs by downregulating CSE protein expression and directly reacting with H(2)S molecule. Interaction of MG with H(2)S may be one of future directions for the studies on glucose metabolism and the development of insulin resistance syndromes.