The shoot and root cultures of Polygonum tinctorium fed with indole were incubated under 18O2 atmosphere. Indican, indigo, and indirubin were isolated and analyzed by mass spectrometry to assess the oxygen labeling pattern. These three indoxyl derivatives were found to be labeled by 18O. The result suggested that, in P. tinctorium, indole was oxidized by molecular oxygen into indoxyl, which was further converted into indican, indirubin, and indigo.