AbstractFlavones are important secondary metabolites found in many plants. InLoniceraspecies, flavones contribute both physiological and pharmaceutical properties. However, flavone synthase (FNS), the key enzyme responsible for flavone biosynthesis, has not yet been characterized inLoniceraspecies. In this study,FNSIIgenes were identified fromLonicera japonicaThunb. andL. macranthoidesHand.-Mazz. In the presence of NADPH, the recombinant cytochrome P450 proteins encoded byLjFNSII-1.1,LjFNSII-2.1andLmFNSII-1.1converted eriodictyol, naringenin and liquiritigenin to the corresponding flavones directly. The different catalytic properties between LjFNSII-2.1 and LjFNSII-1.1 were caused by a single amino acid substitution at position 242 (glutamic acid to lysine). A methionine at position 206 and a leucine at position 381 contributed considerably to the high catalytic activity of LjFNSII-1.1. In addition, LjFNSII-1.1&2.1 and LmFNSII-1.1 also biosynthesize flavones that were further modified byO-glycosylation in transgenic tobacco. The expression levels of theFNSIIgenes were consistent with flavone accumulation patterns in flower buds. Our findings suggested that the weak catalytic activity of LmFNSII-1.1 and the relatively low expression ofLmFNSII-1.1in flowers might be responsible for the low levels of flavone accumulation in flower buds ofL. macranthoides.