Mitomycin C (MC) is an antitumor antibiotic derived biosynthetically from 3-amino-5-hydroxybenzoic acid (AHBA), D-glucosamine, and carbamoyl phosphate. A gene (mitA) involved in synthesis of AHBA has been identified and found to be linked to the MC resistance locus, mrd, in Streptomyces lavendulae. Nucleotide sequence analysis showed that mitA encodes a 388-amino-acid protein that has 71% identity (80% similarity) with the rifamycin AHBA synthase from Amycolatopsis mediterranei, as well as with two additional AHBA synthases from related ansamycin antibiotic-producing microorganisms. Gene disruption and site-directed mutagenesis of the S. lavendulae chromosomal copy of mitA completely blocked the production of MC. The function of mitA was confirmed by complementation of an S. lavendulae strain containing a K191A mutation in MitA with AHBA. A second gene (mitB) encoding a 272-amino-acid protein (related to a group of glycosyltransferases) was identified immediately downstream of mitA that upon disruption resulted in abrogation of MC synthesis. This work has localized a cluster of key genes that mediate assembly of the unique mitosane class of natural products.