Anthocyanins and flavones are biosynthesized from flavanones as a common intermediate, which is the common metabolic branch point for anthocyanin and flavone biosynthesis. The conversion of dihydroflavonols from flavanones for anthocyanin biosynthesis and the conversion of flavones from flavanone for flavone biosynthesis are catalyzed by flavanone 3-hydroxylase (F3H) and flavone synthase (FNSI, II), respectively. Therefore, to elucidate the molecular genetic mechanism of copigmentation between anthocyanins and flavones, the F3H and FNS genes and their transcriptional factors should be characterized, and in this study, 3 cDNA clones encoding F3H named IhF3H1, IhF3H2, and IhF3H3 were isolated and characterized from the flower buds of Dutch iris, Iris × hollandica. Nucleotide sequence analysis showed that IhF3H1 (Genbank accession no. AB183826) is 1,313 bp long and contains an open reading frame (ORF) encoding 370 amino acids with a calculated molecular mass of 40,995 Da and an isoelectric point (pI) of 5.47. IhF3H2 (Genbank accession no. AB265225) is 1,295 bp long and contains an ORF encoding 372 amino acids with a calculated molecular mass of 41,139 Da and a pI of 6.42. IhF3H3 (Genbank accession no. AB265226) is 1,335 bp long and contains an ORF encoding 372 amino acids with a calculated molecular mass of 41,117 Da and a pI of 5.69. The soluble crude protein extracts of Escherichia coli cells expressing IhF3H1, IhF3H2, and IhF3H3 were subjected to flavanone 3-hydroxylation assays in the presence of naringenin as a substrate and 2-oxoglutarate, ascorbate, and FeSO₄ as cofactors. Heterologous expression demonstrated that each IhF3H cDNA encodes functional flavanone 3-hydroxylase, which catalyzes 3-hydroxylation from naringenin to dihydroflavonol.