Abstract Escherichia coli cells growing on glycolate as a sole source of carbon synthesise an enzyme which catalyses the oxidation of this compound to glyoxylate. The formation of glyoxylate from glycolate by extracts of such cells is dependent upon the presence of the artificial electron acceptors phenazine methosulphate and 2,6-dichlorophenolindophenol. The enzyme catalysing this conversion has been partially purified from cell extracts. The enzyme readily oxidised glycolate (Km 4·10−5M) and d (—)-lactate (Km 7·10−4M, but l (+)-lactate was oxidised only slowly. Maximum rates of glycolate oxidation were observed in the pH range 8.0–8.8. The enzyme was relatively insensitive to metal complexing agents but was sensitive to sulphydryl inhibitors. Addition of glycolate to the enzyme produced spectral changes characteristic of the reduction of flavin and cytochrome components, but no cofactors have been positively identified.