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Abstract: Flavonoid 3′-hydroxylase (F3′H) and flavonoid 3′,5′-hydroxylase (F3′5′H) are cytochrome P450 enzymes and determine the B-ring hydroxylation pattern of flavonoids by introducing hydroxyl groups at the 3′- or the 3′- and 5′-position, respectively. Sequence identity between F3′H and F3′5′H is generally low since their divergence took place early in the evolution of higher plants. However, in the Asteraceae the family-specific evolution of an F3′5′H from an F3′H precursor occurred, and consequently sequence identity is substantially higher. We used this phenomenon for alignment studies, in order to identify regions which could be involved in determining substrate specificity and functionality. Subsequent construction and expression of chimeric genes indicated that substrate specificity of F3′H and F3′5′H is determined near the N-terminal end and the functional difference between these two enzymes near the C-terminal end. The impact on function of individual amino acids located in substrate recognition site 6 (SRS6) was further tested by site-directed mutagenesis. Most interestingly, a conservative Thr to Ser exchange at position 487 conferred additional 5′-hydroxylation activity to recombinant Gerbera hybrida F3′H, whereas the reverse substitution transformed recombinant Osteospermum hybrida F3′5′H into an F3′H with low remaining 5′-hydroxylation activity. Since the physicochemical properties of Thr and Ser are highly similar, the difference in size appears to be the main factor contributing to functional difference. The results further suggest that relatively few amino acids exchanges were required for the evolutionary extension of 3′- to 3′,5′-hydroxylation activity. [Copyright &y& Elsevier]