Aklavinone is an aglycone of aclacinomycin A which is an important antitumor drug. Genes for the biosynthesis of aklavinone were cloned from Streptomyces galilaeus 3AR-33, an aklavinoneproducing mutant, by use of the actI and actIII polyketide synthase gene probes.Restriction mapping and Southern analysis of the DNA cloned in a λ phage vector established that the DNA represented three different regions of the S. galilaeus 3AR-33 genome that contained 3.4, 2.5, and 4.1kb BamHI fragments which hybridized with actIII. Of those, only the 3.4kb fragment also hybridized with actl. Complementation experiments with specifically blocked mutants confirmed that the cloned 3.4kb BamHI fragment contains the genes required for the early stage of polyketide synthesis in aklavinone biosynthesis.