Flavonoids are ubiquitous secondary plant metabolites which function as protectants against UV light and pathogens and are involved in the attraction of pollinators as well as seed and fruit dispersers. The hydroxylation pattern of the B-ring of flavonoids is determined by the activity of two members of the vast and versatile cytochrome P450 protein (P450) family, the flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H). Phylogenetic analysis of known sequences of F3'H and F3'5'H indicated that F3'5'H was recruited from F3'H before the divergence of angiosperms and gymnosperms. Seven cDNAs were isolated from species of the Asteraceae family, all of which were predicted to code for F3'Hs based on their sequences. The recombinant proteins of four of the heterologously in yeast expressed cDNAs exhibited the expected F3'H activity but surprisingly, three recombinant proteins showed F3'5'H activity. Phylogenetic analyses indicated the independent evolution of an Asteraceae-specific F3'5'H. Furthermore, sequence analysis of these unusual F3'5'H cDNAs revealed an elevated rate of nonsynonymous substitutions as typically found for duplicated genes acquiring new functions. Since F3'5'H is necessary for the synthesis of 3',4',5'-hydroxylated delphinidin-derivatives, which normally provide the basis for purple to blue flower colours, the evolution of an Asteraceae-specific F3'5'H probably reflects the adaptive value of efficient attraction of insect pollinators.