Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3beta-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid "C ring"; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate. Here we describe work on ANS, FLS, and FHT utilizing several different substrates carried out in 18O2/16OH2, 16O2/18OH2, and 18O2/18OH2 atmospheres. In the 18O2/16OH2 atmosphere close to complete incorporation of a single 18O label was observed in the dihydroflavonol products (e.g. (2R,3R)-trans-dihydrokaempferol) from incubations of flavanones (e.g. (2S)naringenin) with FHT, ANS, and FLS. This and other evidence supports the intermediacy of a reactive oxidizing species, the oxygen of which does not exchange with that of water. In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R,3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzyme-bound C-3 ketone/3,3-gem-diol intermediate.