In this study, administration of 5-hydroxy-L-tryptophan to growing cells yielded a new blue pigment, oxyviolacein, whose chemical structure was determined to be 5-(5-hydroxyindol-3-yl)-3-(5-hydroxy-3-oxoindolylidene)-4 pyrroline-2-one mainly via proton- and 13C-NMR. Feeding experiments with deuterium and 13C-labeled 5-hydroxytryptophan unambiguously demonstrated that tryptophan hydroxylation is the first step in violacein biosynthesis. No incorporation of 5-hydroxy-[4,6-2H2]tryptamine indicated decarboxylation of 5-hydroxy-L-tryptophan occurs at a later stage, not the second step. Lack of incorporation of 5-hydroxy-[4,6-2H2]indole suggested tryptophanase is unlikely involved. Previous work elucidated that violacein skeletons derive exclusively from tryptophan (all C, N, H) and molecular oxygen (O), with indole nucleus migration in one tryptophan molecule. Here, definitive evidence for 5-hydroxytryptophan as an intermediate was provided using stable-isotopically labeled 5-hydroxytryptophan. The D-form of 5-hydroxytryptophan was inactive for violacein formation, and oxyviolacein was produced only by feeding 5-hydroxytryptophan.