Streptomyces galilaeus ATCC 31133 and ATCC 31671, producers of the anthracyclines aclacinomycin A and 2-hydroxyaklavinone, respectively, formed an anthraquinone, aloesaponarin II, when they were transformed with DNA from Streptomyces coelicolor containing four genetic loci, actI, actIll, actlV, and actVII, encoding early reactions in the actinorhodin biosynthesis pathway. Subcloning experiments indicated that a 2.8-kilobasepair XhoI fragment containing only the actl and actVII loci was necessary for aloesaponarin II for the actll gene, which is the reduction of the keto group at C-9 from the carboxy terminus of the assembled polyketide to the corresponding secondary alcohol. In the presence of the actIll gene, anthraquinones or anthracyclines formed as a result of dehydration and aromatization lack an oxygen function on the carbon on which the keto reductase operated. When S. galiaeus ATCC 31671 was transformed with the DNA carrying the actl, actVII, and actlV loci, the recombinant strain produced two novel anthraquinones, desoxyerythrolaccin, the 3-hydroxy analog of aloesaponarin II, and 1-0-methyldesoxyerythrolaccin. The results obtained in these experiments together with earlier data suggest a pathway for the biosynthesis of actinorhodin and related compounds by S. coelicolor.The biosynthesis of the benzoisochromanequinone antibiotic actinorhodin (4), a polyketide antibiotic produced by Streptomyces coelicolor, has been studied intensively in recent years from both the biochemical (7,13,14) and genetic (15,(22)(23)(24)30) viewpoints. Seven classes of blocked mutants have been described and placed in sequential order on the basis of cosynthetic and chemical studies (7,13,30). Studies on the molecular genetics of actinorhodin biosynthesis have provided fundamental knowledge on the structure and organization of antibiotic biosynthesis genes in streptomycetes, including the clustering of antibiotic biosynthesis structural genes (23,24), production of the antibiotic by recombinant strains carrying all of the biosynthesis genes on a plasmid (23), and the potential homology shared by certain structural genes within several different pathways (i.e., the genes encoding polyketide synthases [22]). Actinorhodin