The role of the glyoxalase system in the detoxification of a-ketoaldehydes has made it an interesting system for the development of chemotherapeutic agents. It has been shown that inhibitors of glyoxalase I, the first enzyme in the pathway, have the ability to inhibit murine tumor cell growth in vivo. Tumor toxicity is attributed to an increase in intracellular concentrations of methylglyoxal, which has the ability to form DNA adducts and prevent DNA synthesis. A new class of compound is presented here, which has the ability to covalently modify the active site of human glyoxalase I (hGlxI). These compounds were predicted to be complete irreversible inhibitors of hGlxI, but the current studies show that irreversible inhibition is limited and the enzyme is able to maintain a fraction of its original catalytic activity. The data presented suggest that it is possible that these compounds covalently modify only one active site of hGlxI and that the other active site maintains a fraction of its catalytic activity. Mass spectrometry experiments show that Cys60 is the site of covalent modification. These compounds are the first reported to have the ability to covalently modify a residue in the active site of hGlxI. Although these molecules are not complete irreversible inhibitors of hGlxI, they could possibly serve as templates for the development of new compounds that may have improved potency.