The antibiotic compound pyrroindomycin B contains an indole ring chlorinated in the 5 position. The indole ring is probably derived from tryptophan, and thus primers derived from conserved regions of tryptophan halogenases were used to amplify and clone a DNA fragment that was then used to isolate a tryptophan 5-halogenase gene (pyrH) from a cosmid library of the pyrroindomycin producer Streptomyces rugosporus LL-42D005. A gene disruption mutant in the tryptophan 5-halogenase gene no longer produced pyrroindomycin B, but still produced pyrroindomycin A, the nonhalogenated derivative. The halogenase gene could be overexpressed in Pseudomonas fluorescens BL915 DeltaORF1 and was purified to homogeneity by immobilized metal chelate ion affinity chromatography. Chlorinating and brominating activities with tryptophan as a substrate were detected in cell-free extracts and for the purified enzyme.