The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3'-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3',4'-dihydroxyphenyllactate and the 3'-hydroxylation of caffeoyl-4'-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K (m)-values for 4-coumaroyl-3',4'-dihydroxyphenyllactate and caffeoyl-4'-hydroxyphenyllactate were determined to be at 5 microM and 40 microM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.