<jats:title>ABSTRACT</jats:title> <jats:p> <jats:italic>Streptomyces ambofaciens</jats:italic> synthesizes the macrolide antibiotic spiramycin. The biosynthetic gene cluster for spiramycin has been characterized for <jats:italic>S. ambofaciens.</jats:italic> In addition to the regulatory gene <jats:italic>srmR</jats:italic> ( <jats:italic>srm22</jats:italic> ), previously identified (M. Geistlich et al., Mol. Microbiol. 6:2019-2029, 1992), three putative regulatory genes had been identified by sequence analysis. Gene expression analysis and gene inactivation experiments showed that only one of these three genes, <jats:italic>srm40</jats:italic> , plays a major role in the regulation of spiramycin biosynthesis. The disruption of <jats:italic>srm22</jats:italic> or <jats:italic>srm40</jats:italic> eliminated spiramycin production while their overexpression increased spiramycin production. Expression analysis was performed by reverse transcription-PCR (RT-PCR) for all the genes of the cluster in the wild-type strain and in the <jats:italic>srm22</jats:italic> ( <jats:italic>srmR</jats:italic> ) and <jats:italic>srm40</jats:italic> deletion mutants. The results from the expression analysis, together with the ones from the complementation experiments, indicated that Srm22 is required for <jats:italic>srm40</jats:italic> expression, Srm40 being a pathway-specific activator that controls most, if not all, of the spiramycin biosynthetic genes.