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Cell cultures of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) are used for the industrial production of the red naphthoquinone pigment shikonin. p-Hydroxybenzoic acid (PHB), derived from cinnamic acid, is a key intermediate in the biosynthetic pathway leading to shikonin (1). Based on former studies in L. erythrorhizon, Yazaki et al. (2) proposed p-hydroxybenzaldehyde (PHAL) as an intermediate of the in vitro formation of PHB from p-coumaric acid (PCA). Recent in vitro experiments with E14CJPCA and [14C]PCA-C0A under improved assay conditions clearly demonstrate that PCA-CoA rather than PHAL is the main intermediate of PHB biosynthesis. The formation of PHB from PCA is dependent on the cofactors ATP, Mg, coenzyme A, and NAD. Activity of 4-coumarate: CoA-ligase, forming PCA-CoA from PCA, could be detected. ATP, Mg and CoA are the essential cofactors for this reaction. PCA-CoA is converted rapidly to PHB under in vitro conditions. NAD is the only essential cofactor for this reaction, which is a direct conversion of the CoA ester and does not proceed via free PCA or free PHAL.