<jats:title>ABSTRACT</jats:title> <jats:p> Sequencing of a 4.3-kb DNA region from the chromosome of <jats:italic>Streptomyces argillaceus</jats:italic> , a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one ( <jats:italic>orfA</jats:italic> ) codes for a protein that resembles several transport proteins. The second one ( <jats:italic>mtmR</jats:italic> ) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the <jats:italic>Streptomyces</jats:italic> antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the <jats:italic>mtmR</jats:italic> region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of <jats:italic>mtmR</jats:italic> in a high-copy-number vector in <jats:italic>S. argillaceus</jats:italic> caused a 16-fold increase in mithramycin production. The <jats:italic>mtmR</jats:italic> gene restored actinorhodin production in <jats:italic>Streptomyces coelicolor</jats:italic> JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by <jats:italic>Streptomyces lividans</jats:italic> TK21. A 241-bp region located 1.9 kb upstream of <jats:italic>mtmR</jats:italic> was found to be repeated approximately 50 kb downstream of <jats:italic>mtmR</jats:italic> at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by <jats:italic>S. argillaceus</jats:italic> is proposed.