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Ansamitocin P-3 (AP-3) and its derivatives were reported to inhibit the growth of various leukemia cell lines and human solid tumors [1-3].Although there is a demand for large and cheap supply of AP-3 for synthesis of its derivatives,its production level is still low.No information is yet available regarding the gene transcriptional level and enzymatic activity during AP-3 fermentation.In this work.supply of ester side chain (by isobutanol addition) to enhance AP-3 production by Actinosynnema pretiosum was investigated with simultaneous analysis of transcriptional levels of AP-3 biosynthetic genes as well as enzymatic activities related to precursors utilization.