Naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 incorporated dioxygen at the C7 and C8 positions on the A-rings of flavone and isoflavone with different stereoselectivity, resulting in the formation of (7S,8S)-dihydroxy-2-phenyl-7,8-dihydro-4H-chromen-4-one (flavone-cis-(7S,8S)-dihydrodiol) and (7R,8R)-dihydroxy-3-phenyl-7,8-dihydro-4H-chromen-4-one (isoflavone-cis-(7R,8R)-dihydrodiol), respectively. In addition, NDO was shown to incorporate dioxygen at the C5 and C6 positions on the A-ring and the C2' and C3' positions on the B-ring of isoflavone, resulting in the production of (5S,6R)-dihydroxy-3-phenyl-5,6-dihydro-4H-chromen-4-one (isoflavone-cis-(5S,6R)-dihydrodiol) and 3-[(5S,6R)-5,6-dihydroxycyclohexa-1,3-dienyl]-4H-chromen-4-one (isoflavone-cis-(2'R,3'S)-dihydrodiol), respectively. The metabolites were identified by LC/MS, H-1, and C-13 NMR analyses and TD-SCF calculations combined with CD spectroscopy. In the case of flavone biotransformation, formation of flavone-(7S,8S)-dihydrodiol is likely to be the result of hydrogen bond interactions between the substrate and the active site of the dioxygenase. On the contrary, regioselective dioxygenation of isoflavone was found not to occur, and this may be due to the fact that the same hydrogen bonds that occur in the case of the flavone reaction cannot be established due to steric hindrance caused by the position of the B-ring. It is therefore proposed that the regioselectivity and stereoselectivity of NDO from strain NCIB 9816-4 are controlled by the position of the phenyl ring on flavone molecules.