Abstract: Normal and reversed-phase chromatographic methods have been systematically investigated for separating spiramycin(SPM) and acetylspiramycin (Ac-SPM). The four components of Ac-SPM have been well separated by HPLC using μ-Bondapak C₁₈ column with a mobile phase composed of 0.01 mol/L H₃PO₄-CH₃OH (55:45). Two chromatographic systems were chosen to estimate the three components of SPM. In the first system 0.01 mol/L H₃PO₄—CH₃OH (60:40) eluent is used, and in the second system Zorbax CN column with methanol—isopropyl alcohol—cyclohexane (5:6:89) mobile phase is employed. It has been found that the contents of each component in the SPM made in China are different from those made in Japan, namely, in the Chinese SPM, SPM Ⅱ and Ⅲ are the major components, but in Japanese SPM, SPM Ⅰ is the chief component. Moreover, in the Chinese Ac-SPM, diacetyl spiramycin Ⅱ, Ⅲ are dominant, while in the Japanese Ac-SPM, di-and monoacetyl spiramycin Ⅱ are the major components.