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The biosynthesis of novobiocin was investigated using isotopically labelled compounds. The amino‐group of the coumarin unit was found to be derived from tyrosine, other typical 7‐oxycoumarin precursors were not incorporated into the coumarin unit of novobiocin, 4′‐hydroxyphenylpyruvate was a better precursor of the substituted benzoic acid unit than was 4′‐hydroxycinnamic acid. A biosynthetic route to this unit of novobiocin is suggested. Synthetic pathways for 2′,4′‐dihydroxyphenylalanine‐1‐14C and 4‐hydroxybenzaldehyde‐U‐14C are reported for the first time.

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