Although but weak inhibitors of glyoxalase I under steady-state conditions, flavins are reduced by yeast glyoxalase I (lactoyl-glutathione lyase, EC 4.4.1.5) plus its substrate (the hemithiolacetal from glutathione and phenylglyoxal) during catalytic turnover. Studies with 10-ethylisoalloxazine showed that this flavin reduction was peculiar not merely to glyoxalase I's substrate, but was characteristic of the complete system, enzyme plus substrate undergoing catalytic turnover. Flavins are poor hydride-ion acceptors and the reduction observed most likely represents an oxidative trap of a transient carbanion formed in the glyoxalase I mechanism of action. Hydrophobic flavins were more efficient traps than the hydrophilic ones, and values of the Km for the phenylglyoxal: glutathione hemithiolacetal adduct measured by the flavin-reaction and by normal steady-state kinetics were closely similar. This argues that trapping has occurred of an enediolate ion (an enzyme-generated carbanion) still bound to glyoxalase I.