During genetic study on obtaining high-yield variants of aclacinomycin A-producer, a variety of blocked mutants were isolated from Streptomyces galilaeus MA144-M1 and were characterized. The isolated mutants included those which accumulated only specific components of parental glycosides, those which produced new aklavinone glycosides devoid of parental rhodosamine or both rhodinose and cinerulose, those which produced non-glycosidic aglycones, and antibiotic-negative mutants, some of which were able to glycosidate exogenous aklavinone. By biotransformation with the aglycone feeding culture, the precursor activity of new aglycones was also tested. From the results and in relation to the characterization of isolated mutants, the biosynthetic pathway of aclacinomycin A and related antibiotics is discussed.