The biosynthesis of epoxypseudoisoeugenol-2-methylbutyrate (EPB), a rare phenylpropanoid of the genus Pimpinella, was investigated in vitro by means of a leaf-differentiating callus culture of Pimpinella anisum. In an effort to corroborate an earlier proposed biosynthetic pathway of EPB, the step between p-coumaryl alcohol and (E)-anethole was reinvestigated. Further feeding experiments with 1 4 C-labelled precursors and preliminary studies at the enzyme level clearly revealed that anol, and not p-methoxycinnamyl alcohol as previously asserted, is an obligatory intermediate in EPB biosynthesis. S-Adenosyl-l-methionine: anol-O-methyltransferase, a key enzyme in EPB biosynthesis, was demonstrated and characterized for the first time.