In contrast to the well-studied B-ring hydroxylation of several flavonoid classes, knowledge on the establishment of the B-ring hydroxylation pattern in chalcones is still limited. Previous studies using enzyme preparations from Dahlia variabilis petals showed that a NADPH-dependent, membrane bound enzyme is responsible, but the possible involvement of the common flavonoid 3'-hydroxylase (F3'H) remained unclear. Therefore, we tested for the first time recombinant F3'Hs from eleven ornamental plant species, mostly Asteraceae, for their acceptance of chalcones as substrates. Ten of the 11 enzymes tested were not able to hydroxylate the 6'-deoxychalcone isoliquiritigenin at position 3, indicating the involvement of a specific enzyme in the hydroxylation of chalcones in ring B. Recombinant F3'H from Tagetes erecta accepted isoliquiritigenin as a substrate but low conversion rates and kinetic data clearly indicate that flavonoids are the preferred substrates. As microsomal preparations from Tagetes erecta petals do not hydroxylate chalcones at position 3, the observed low chalcone 3-hydroxylase activity of recombinant Tagetes F3'H seems to be a result of the use of a heterologous overexpression system. Cytochrome P450 reductase-specific antibodies provided final evidence that the CH3H reaction is catalyzed by a cytochrome-P450-dependent monooxygenase. This supports future cloning strategies based on respective conserved regions. (C) 2009 Elsevier Ireland Ltd. All rights reserved.