Coumarins are derived via the phenylpropanoid pathway in plants. The 2H-1-benzopyran-2-one core structure of coumarins is formed via the ortho-hydroxylation of cinnamates, trans/cis isomerization of the side chain, and lactonization. Ortho-hydroxylation is a key step in coumarin biosynthesis as a branch point from lignin biosynthesis; however, ortho-hydroxylation of cinnamates is not yet fully understood. In this study, scopoletin biosynthesis was explored using Arabidopsis thaliana, which accumulates scopoletin and its beta-glucopyranoside scopolin in its roots. T-DNA insertion mutants of caffeoyl CoA O-methyltransferase 1 (CCoAOMT1) showed significant reduction in scopoletin and scopolin levels in the roots, and recombinant CCoAOMT1 exhibited 3'-O-methyltransferase activity on caffeoyl CoA to feruloyl CoA. These results suggest that feruloyl CoA is a key precursor in scopoletin biosynthesis. Ortho-hydroxylases of cinnamates were explored in the oxygenase families in A. thaliana, and one of the candidate genes in the Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2OGD) family was designated as F6'H1. T-DNA insertion mutants of F6'H1 showed severe reductions in scopoletin and scopolin levels in the roots. The pattern of F6'H1 expression is consistent with the patterns of scopoletin and scopolin accumulation. The recombinant F6'H1 protein exhibited ortho-hydroxylase activity for feruloyl CoA (K(m) = 36.0 +/- 4.27 microM; k(cat) = 11.0 +/- 0.45 sec(-1)) to form 6'-hydroxyferuloyl CoA, but did not hydroxylate ferulic acid. These results indicate that Fe(II)- and 2-oxoglutarate-dependent dioxygenase is the pivotal enzyme in the ortho-hydroxylation of feruloyl CoA in scopoletin biosynthesis.