<jats:title>ABSTRACT</jats:title> <jats:p> The biosynthetic gene cluster for the aromatic polyketide steffimycin of the anthracycline family has been cloned and characterized from “ <jats:italic>Streptomyces steffisburgensis</jats:italic> ” NRRL 3193. Sequence analysis of a 42.8-kbp DNA region revealed the presence of 36 open reading frames (ORFs) (one of them incomplete), 24 of which, spanning 26.5 kb, are probably involved in steffimycin biosynthesis. They code for all the activities required for polyketide biosynthesis, tailoring, regulation, and resistance but show no evidence of genes involved in <jats:sc>l</jats:sc> -rhamnose biosynthesis. The involvement of the cluster in steffimycin biosynthesis was confirmed by expression of a region of about 15 kb containing 15 ORFS, 11 of them forming part of the cluster, in the heterologous host <jats:italic>Streptomyces albus</jats:italic> , allowing the isolation of a biosynthetic intermediate. In addition, the expression in <jats:italic>S. albus</jats:italic> of the entire cluster, contained in a region of 34.8 kb, combined with the expression of plasmid pRHAM, directing the biosynthesis of <jats:sc>l</jats:sc> -rhamnose, led to the production of steffimycin. Inactivation of the <jats:italic>stfX</jats:italic> gene, coding for a putative cyclase, revealed that this enzymatic activity participates in the cyclization of the fourth ring, making the final steps in the biosynthesis of the steffimycin aglycon similar to those in the biosynthesis of jadomycin or rabelomycin. Inactivation of the <jats:italic>stfG</jats:italic> gene, coding for a putative glycosyltransferase involved in the attachment of <jats:sc>l</jats:sc> -rhamnose, allowed the production of a new compound corresponding to the steffimycin aglycon compound also observed in <jats:italic>S. albus</jats:italic> upon expression of the entire cluster.