The effects of glucose, butyrate, and cerulenin on the formation of spiramycin I 3-hydroxyl acylase were investigated by using the cell-free extract prepared from the spiramycin-producing strain of Streptomyces ambofaciens KA-1028. Glucose induced the formation of the acylase, and this induction was remarkably repressed by butyrate. Cerulenin, on the other hand, not only cancelled the repression by butyrate but also stimulated the formation of the acylase. The unsuccessful trapping of spiramycin I as an intermediate during the bioconversion from neospiramycin I to spiramycin III in the presence of cerulenin was due to the rapid acylation of spiramycin I caused by the acylase induced by cerulenin.