Contradictory results in the literature concerning the role of L-tryptophan in the biosynthesis of the indigo precursors, indican and isatan B, in four higher plants belonging to four plant families have been resolved. [3-¹³C]- Indole and L-[3-¹³C]-tryptophan were synthesized by standard techniques. Feeding experiments with the labelled precursors unequivocally showed by ¹³C NMR and mass spectroscopy, that indole and not L-tryptophan is the biosynthetic precursor for the indoxyl derivatives found in plants.