We have isolated a cDNA clone that corresponds to the Ht1 locus of petunia which controls the hydroxylation of dihydrokaempferol to dihydroquercetin and of naringenin to eriodictyol by the action of flavonoid 3'-hydroxylase (F3'H). The cDNA encodes a 512 amino acid polypeptide with regions of similarity to petunia flavonoid 3',5'-hydroxylases (F3'5'H). Both F3'H and F3'5'H are cytochromes P450 and are key enzymes in the flavonoid pathway leading to the production of the coloured anthocyanins. The F3 'H transcript is most abundant in petals from flowers at an early stage of development and levels decline as the flower matures. Transcripts are also detected in the ovaries, sepals, peduncles, stems and anthers of the petunia plant. No or very reduced levels of transcripts are detected in ht1/ht1 lines. This is the first report of isolation of a F3 'H cDNA clone from any species.