The yellow coloration of snapdragon (Antirrhinum majus) flowers is mainly provided by the 6-glucosides of aureusidin and bracteatin. However, the biochemical mechanism of aurone biosynthesis is not well understood. In this study, we have identified aurone-biosynthesizing activity in the extracts of yellow snapdragon flowers. Incubation of 2',4',6',4-tetrahydroxychalcone (THC) with an enzyme preparation in the presence of H(2)O(2) caused the enzymatic formation of a single product, aureusidin, without the formation of a previously proposed 2-(alpha-hydroxybenzyl)coumaranone intermediate. The formation of aureusidin from THC was specifically observed with yellow flowers as well as aurone-accumulating flowers of other colors. The pH optimum for the enzymatic formation of aureusidin was around 5.4. Stoichiometric studies showed that one mole of aureusidin formation was accompanied by the consumption of one mole of oxygen with no detectable consumption of H(2)O(2), which may work as an enzyme activator. The oxidative formation of aureusidin from THC could be explained in terms of the action of a single enzyme, an internal monooxygenase catalyzing the 3-hydroxylation and oxidative cyclization of THC. Incubation of 2',4',6',3,4-pentahydroxychalcone (PHC) with an enzyme yielded both aureusidin and bracteatin at an approximate molar ratio of 6:1. In this case, H(2)O(2) was not required for enzyme activity but rather inhibited the reaction. The 4'-glucosides of THC and PHC could also act as substrates for the formation of the 6-glucosides of aurones. These results suggest that aureusidin can be produced from either THC or PHC, whereas bracteatin is not produced through the 5'-hydroxylation of aureusidin but arise solely from PHC.