Rebeccamycin, a halogenated natural product of the indolocarbazole family, is produced by Saccharothrix aerocolonigenes ATCC39243. Several rebeccamycin analogues, which target DNA topoisomerase I or II, have already entered clinical trials as anticancer drugs. Using as a probe an internal fragment of ngt, a Saccharothrix aerocolonigenes gene encoding an indolocarbazole N-glycosyltransferase, we isolated a DNA region that directed the biosynthesis of rebeccamycin when introduced into Streptomyces albus. Sequence analysis of 25.6 kb revealed genes for indolocarbazole core formation, halogenation, glycosylation, and sugar methylation, as well as a regulatory gene and two resistance/secretion genes.