An enzyme preparation able to acylate the hydroxyl group at C-3 of the lactone ring of spiramycin was obtained from the spiramycin-producing strain, Streptomyces ambofaciens ISP-5053. The enzyme was purified about 33-fold from the crude extract by means of ammonium sulfate fractionation, diethylaminoethyl (DEAE) cellulose batchwise elution and DEAE cellulose column chromatography. The optimum pH for the enzyme activity was 8.5. The enzyme was activated by Ca1+ , Mg1+ , and MnI+ in this order, but was inhibited by various SH reagents. Spiramycin I was the best substrate for the enzyme. The enzyme showed no preference between acetyl-CoA and propionyl-CoA.