The shoot and root cultures of Polygonum tinctorium fed with indole were incubated under $^{18}O_2$ 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 $^{18}O$. The result suggested that, in P. tinctorium, indole was oxidized by molecular oxygen into indoxyl, which was further converted into indican, indirubin, and indigo.