Abstract: Thin-layer chromatography was used for separation of spiramycin (SPM) and acetylspiramycin (Ac-SPM). 0.3 mol/L NaOH in 0.5% CMC-Na solution was selected to spread plates with silica gel (3.3:1), methanol—ethyl acetatecyclohexane (10:45:45) and (8:46:46) were employed for separating SPM and AcSPM respectively. It has been found that the content of each component in SPM made in China is different from that made in Japan, namely, in the former SPM Ⅱ, Ⅲ are the chief components, but in the latter SPM, SPM Ⅰ is the chief one. Moreover, in Ac-SPM made in China, diacetylspiramycin Ⅱ, Ⅲ are dominant and in Ac-SPM made in Japan, di- and monoacetylspiramycin Ⅱ are the principal components.