Dihydroxyacetanilide, 4, is oxidized to the SR,6S-epoxyquinone, 3, by dihydroxyacetanilide epoxidase-I (DHAE I) from Streptomyces LL-C10037, without the assistance of an organic cofactor. C-13 NMR analysis revealed that in the presence of O-18(2) a full equivalent of O-18 is incorporated at the epoxide. However, control reactions revealed the rapid exchange of the C-4 carbonyl with (H2O)-O-18. By coupling the DHAE I reaction with 2-acetamido-5,6-epoxy-1,4-benzoquinone oxidoreductase (AEBQOR I) from the same organism, NADP, and an NADPH regeneration system based on glucose 6-phosphate dehydrogenase, the epoxyquinol LL-C10037 alpha, 1, was produced with similar to 20% incorporation of a second O-18 atom at the C-4 alcohol. Therefore, DHAE I is a dioxygenase with an epoxidation mechanism essentially the same as has been observed for the dihydrovitamin K epoxidation occurring during the mammalian vitamin K-dependent glutamate carboxylase reaction.