<jats:title>ABSTRACT</jats:title> <jats:p> The biosynthetic gene cluster of the aminocoumarin antibiotic coumermycin A <jats:sub>1</jats:sub> was cloned by screening of a cosmid library of <jats:italic>Streptomyces rishiriensis</jats:italic> DSM 40489 with heterologous probes from a dTDP-glucose 4,6-dehydratase gene, involved in deoxysugar biosynthesis, and from the aminocoumarin resistance gyrase gene <jats:italic>gyrB</jats:italic> <jats:sup>r</jats:sup> . Sequence analysis of a 30.8-kb region upstream of <jats:italic>gyrB</jats:italic> <jats:sup>r</jats:sup> revealed the presence of 28 complete open reading frames (ORFs). Fifteen of the identified ORFs showed, on average, 84% identity to corresponding ORFs in the biosynthetic gene cluster of novobiocin, another aminocoumarin antibiotic. Possible functions of 17 ORFs in the biosynthesis of coumermycin A <jats:sub>1</jats:sub> could be assigned by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by an insertional gene inactivation experiment, which resulted in an abolishment of coumermycin A <jats:sub>1</jats:sub> production.