Germinated spores of Actinomadura verrucosospora subsp. neohibisca E-40, a high pradimicins producer, were mutagenized by N-methyl-N'-nitro-N-nitrosoguanidine and/or UV treatment. Thirty-seven mutants which did not produce pradimicin were selected to test for cosynthesis ability, and classified into nine classes. On the basis of their cosynthesis ability and bioconversion results, we concluded that strain JN-213 (class III) was a true converter and that strains JN-219 (class IV), JN-47 (class V) and JNU-46 (class VI) were secretors accumulating biosynthetic intermediates of pradimicin, and that strains JN-59 (class VII), JN-58 (class VIII) and JN-207 (class IX) were producers of shunt metabolites of pradimicin biosynthesis. TLC and HPLC analyses of the fermentation broths of individual strains showed that 8 new compounds were produced along with pradinone I, pradimicinone I, 11-O-demethylpradimicinone II and 7-O-methylpradimicinone II.