Spiramycin and midecamycin are 16-membered macrolide antibiotics with very similar chemical structures. Spiramycin has three components, namely spiramycin I, II and III. Spiramycin II and III are, respectively, the O-acetyl and propionyl derivatives at C3-hydroxyl group of spiramycin I. Midecamycin has four components, and the C3-hydroxyl group of midecamycin is all O-propionylated. The enzyme adding acyl group(s) at the C3-hydroxyl group during the biosynthesis of spiramycin and midecamycin is 3-O-acyltransferase. The 3-O-acyltransferases for spiramycin and midecamycin are also very similar, and presume to function when exchanged. To explore whether the 3-O-acyltransferase for midecamycin biosynthesis hold still the character of selective and efficient propionylation for spiramycin I at its C3-hydroxyl group, we inserted mdmB, the 3-O-acyltransferase gene from Streptomyces mycarofaciens ATCC 21454 for midecamycin biosynthesis, into a mutant strain of S. spiramyceticus F21, in which the 3-O-acyltransferase gene for spiramycin biosynthesis, sspA, was deleted; and the mdmB was integrated exactly into the chromosomal site where the sspA was deleted. We name this "hybrid" strain as SP-mdmB. HPLC analysis of the spiramycin produced by SP-mdmB showed that spiramycin I was still the major component, although the relative proportions of both spiramycin II and III increased significantly. We thus conclude that MdmB from Streptomyces mycarofaciens ATCC 21454 for midecamyicn biosynthesis do not hold the character of selective and efficient propionylation for spiramycin I within S. spiramyceticus F21, and this character is possibly limited in Streptomyces mycarofaciens ATCC 21454 for midecamycin biosynthesis.