Aklavinone (2) is an aglycone of aclacinomycin A (1) which is an important antitumor drug. Genes for the biosynthesis of aklavinone were cloned from Streptomyces galilaeus 3AR-33, an aklavinone-producing 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 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 hybridized with actI. 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. A 5.5kb region including 3.4kb BamHI fragment of the S. galilaeus 3AR-33 aklavinone gene cluster was sequenced. FRAME analysis revealed the presence of five possible open reading frames (ORFs). Four ORFs (aknX, aknB, aknC and aknD) run divergently from the adjacent aknA gene. The deduced gene product of aknA is homologous to known oxidoreductases. The predicted aknB and aknC gene products are homologous to β-ketoacylsynthses of fatty acid synthases. The aknD gene product is homologous to acyl carrier proteins. The aknX product does not resemble any other known proteins. These results show the aklavinone polyketide synthetase is a multienzyme complex which consists of separate polypeptides rather than the multifunctional polypeptides for the macrolide polyketide synthases.