The biosynthetic pathway of daunomycin (DM) from intermediate aglycone aklavinone (AKN) is studied by biosynthetic conversion of various precursors using non-DM-producing blocked mutants. DM formation starts by (i) 11-oxidation of AKN to yield epsilon-rhodomycinone (epsilon-RMN) and is then achieved by at least six biosynthetic steps in the following order; (ii) 7-O-daunosaminylation of epsilon-RMN to yield D788-6 (5A : 7-O-daunosaminyl epsilon-RMN); (iii) 10-demethylesterification to yield D788-1 (RP : 10-carboxy-13-deoxocarminomycin); (iv) 10-decarboxylation to yield D788-11 (5B : 13-deoxocarminomycin); (v) 4-O-methylation to yield feudomycin A (13-deoxodaunomycin); (vi) 13-oxidation to yield 13-dihydrodaunomycin; (vii) 13-dehydrogenation to yield DM. However, the DM biosynthetic route via carminomycin is not observed. It is also found that most of these possible biosynthetic enzymes involved in DM production can catalyze some related metabolites other than their practical precursors.