The 200-fold purification of glyoxaiase I (EC 4.4.1.5) from calf liver is described. This enzyme exhibits two marked anomalies in its kinetic behavior. There is an initial lag phase of a few seconds, and the plot of velocity versus enzyme concentration is nonlinear, reaching a plateau value. These features are correlated with the kinetics of formation of the hemimercaptal of glutathione and methylglyoxal, suggesting that this adduct is the true substrate of glyoxaiase I.