enol-Pyruvaldehyde (ePY or 2-hydroxypropenal, O=C(H)-C(OH)=CH2) a transient intermediate in the alkaline decomposition of the triosephosphates to methylglyoxal is now observed by UV and H-1 NMR spectroscopy as the immediate product of the methylglyoxal synthetase (MGS) reaction: dihydroxyacetone-P --> P-i + ePY --> methylglyoxal (MG). Analysis of ePY formed from 1-C-13- and (1R, 3S) -[1,3-H-2]-DHAP establishes the stereochemical course of its formation by MGS. Its rate of ketonization is much too slow to be in the sequence required for the assay of MGS by coupling of the MG produced to glyoxalase I (Glx I): MG + glutathione (GSH) --> (S)-lactylglutathione (D-LG). Instead, ketonization occurs by way of the hemithioacetal (HTA) formed between ePY and GSH, and could be either an enzymatic function of Glx I or occur nonenzymatically at an activated rate. Enzymatic ketonization was ruled out because the methyl group of D-LG formed from specifically labeled ePY is achiral. Chemical ketonization of ePY is activated by general bases, such as acetate, and by thiols such as GSH and 2-mercaptoethanol, which disrupt its stabilizing double bond conjugation as hemithioacetal (HTA) adducts. 2-Mercaptoacetate combines both functions, acting as the HTA adduct of ePY with the appended carboxylate group presumably positioned to promote abstraction of the enol proton and protonation of the enolate carbon at an accelerated rate. in the MGS-Glx I system (dihydroxyacetone-P --> ePY, ePY + GSH --> GS-ePY, GS-ePY --> GS-MG, GS-MG --> D-LG), the nonenzymatic 2nd and 3rd steps describe the catalytic role of GSH in the critical ketonization process and set the stage for its participation in the glyoxalase system.