The biosynthetic pathway of pradimicin S (PRM-S) was investigated by using sinefungin and bioconversion experiments with aglycones of pradimicin A (PRM-A) and Actinomadura spinosa AA0851, a PRM-S producer. Addition of sinefungin to the strain inhibited the formation of 11-O-demethyl-7-O-methylpradinone II (11dM-7M-PNII) as also determined to occur with its addition to the PRM-A producer. In feeding PRM-A aglycone and its analogs to the strain early in PRM-S biosynthesis, good identifications of bioconverted products were obtained by frit-FAB LC/MS as follows: 11-O-demethylpradinone II (11dM-PNII), 11dM-7M-PNII, 11-O-demethylpradinone I (11dM-PNI), 11-O-demethylpradimicinone I (11dM-PMNI) and pradimicinone I (PMNI) were converted to PRM-S. Pradimicin B (PRM-B) and pradimicin L (PRM-L) were converted to PRMs-L and -S and PRM-S, respectively. A biosynthetic pathway for PRM-S is proposed.