The flavoenzyme glycolate oxidase oxidizes glycolic acid to glyoxylate andthe latter, more slowly, to oxalate. It is a member of an FMN-dependentenzyme family that oxidizesL-2-hydroxy acids to keto acids. There has beena controversy concerning the chemical mechanism of substrate oxidation bythese enzymes. Do they proceed by hydride transfer, as observed for NAD-dependent enzymes, or by initial formation of a carbanion that transfers theelectrons to the flavin? The present work describes a comparison of the reac-tivity of glycolate, lactate and trifluorolactate with recombinant human gly-colate oxidase, by means of rapid-kinetics experiments in anaerobiosis. Weshow that trifluorolactate is a substrate for glycolate oxidase, whereas it isknown as an inhibitor for NAD-dependent enzymes, as is trifluoroethanolfor NAD-dependent alcohol dehydrogenases. Unexpectedly, it was observedthat, once reduced, a flavin transfers an electron to an oxidized flavin, so thatthe end species is a flavin semiquinone, whatever the substrate. This phenom-enon has not previously been described for a glycolate oxidase. Altogether,considering that another member of this flavoenzyme family (flavocyto-chromeb2, a lactate dehydrogenase) has also been shown to oxidize trifluoro-lactate (Lederer F et al. (2016) Biochim Biophys Acta 1864, 1215–21), thiswork provides another important piece of evidence which is hardly compati-ble with a hydride transfer mechanism for this flavoenzyme family