The glycylmethyl and glycylethyl esters of glutathione have been synthesized and carefully characterized by both 1H-NMR and tandem FAB mass spectrometry. Contrary to previously published studies, these compounds (as their methylglyoxal-thiohemiacetals) do indeed serve as moderately efficient substrates for yeast glyoxalase I, with kcat values that are approx. 3-fold smaller and Km values that are approx. 3-fold larger than those of the thiohemiacetal formed from glutathione. Product inhibition studies show that the glycylmethyl and glycylethyl esters of (S)-D-lactoylglutathione bind approx. 1.4-fold less tightly to the active site than (S)-D-lactoylglutathione. These observations exclude an essential role for the glycyl-CO2- of substrate in active site binding and catalysis.