For a long time, the antibiotic of lactic acid bacteria had been discussed. There were two or more factors to influence the antimicrobial activity of fermented lactic cultures. It was about ten years ago to identify an antibiotic produced by L. acidophilus. But the investigation of its nature has not been progressed noticibly. Acidophilin, produced by L. acidophilus, was one of these antimicrobial agents of lactic acid bacteria. It could be partially purified by Shahani et al., and some of its natures were shown by them. It was expected as an efficient drug and/or antiseptic of food because it might be nontoxic and non-resistent to human body. So it is a fine object to purify acidophilin and to analyze its chemical natures and biological properties. In this research, acidophilin was purified by improved methods of Shahani``s and Hamdan``s. Lactic acid-free and pure acidophilin was purified by Sephadex and silica gel column chromatographies followed by thin-layer chromatography. For the analysis of the chemical structure, ultra-violet, infrared, and NMR spectra were employed and several crews of its structure could be suggested. Some of chemical properties, pH stability, thermostability, and polarity were also revealed. In vitro test of the antimicrobial activity was carried out, so the antimicrobial susceptiblities against several microorganisms were suggested. Through experiments like these, the properties of acidophilin is not exactly correlated with the results of Shahani``s and Hamdan``s. But this is the first one that pure lactic acid-free acidophlin is researched. It is not acidic and hygroscopic. Its UV absorbance maximum is at 243 nm.