Spiramycin (SP) belongs to the 16-member macrolide antibiotics. It contains three components，namely SPⅠ，SPⅡ and SPⅢ，which differ structurally in the acylation moieties on the C₃ of the lactone. The SPⅠ component contains a hydroxyl group at C₃. SPⅡ, and SPⅢ are formed by further acetylation or propionylation of the C₃of SPⅠ，by the same 3-O-acyltransferase (3-O-AT). The study focused on simplifying spiramycin components. Theoretically，disruption/deletion of the (3-O-AT) gene will reduce/stop the acylation of SPⅠ to SPⅡ and SPⅢ. In this study，degenerated primers were designed according to the conserved regions of 3-O-acyltransferase，MdmB and AcyA in the medicamycin and carbomycin producers of S. mycarofaciens and S. thermotolerans，respectively，and an 878bp DNA fragment was amplified from the spiramycin-producer of S. spiramyceticus F21. Blast analysis of the 878bp DNA fragment suggested that it encoded the 3-O-acyltransferase (3-O-AT，sspA) gene for spiramycin biosynthesis. The flanking regions of this 878bp DNA fragment were then amplified by single-oligonucleotide-nested PCR，and a total of 4.3kb DNA was obtained (3457nt among the 4.3kb fragment was sequenced，and deposited in GenBank DQ642742)，covering the whole putative 3-O-acyltransferase gene，sspA. The sspA was then deleted from the S. spiramyceticus F21 genome by double cross-over homologous recombination，mediated by temperature-sensitive plasmid pKC1139. A comparison was done of the components of spiramycins produced by the sspA-deleted mutant strain with that of the parent strain by HPLC analysis, which showed that sspA-deleted mutant produced SPⅠ (72%)，SPⅡ (18%), and SPⅢ(9.6%)，whereas parent strain produced SPⅠ (7.8%)，SPⅡ (67%), and SPⅢ(25%)，respectively，demonstrating the role of sspA in the acylation of SPⅠ into SPⅡ and SPⅢ. The sspA-deleted mutant strain obtained in this study may be used for the production of SPⅠ，or may serve as a good starter for the construction of spiramycin derivatives.