Streptomyces rimosus is an oxytetracycline (OTC) antibiotic producing bacteria that exhibited activities against gram positive and negative bacteria. OTC is used widely not only in medicine but also in production industry. The antibiotic production of streptomyces covers a very wide range of condition. However, antibiotic producers are particularly fastidious cultivated by proper selection of media such as carbon source. In present study we have optimised conditions of OTC production (Composition of production media, p H, shaking and temperature). The results have been shown that bran barley is the optimal media for OTC production at 28C pH5.8 at 150rpm for 5 days. For antibiotic determination, OTC was extracted with different organic solvent. Thin-layer chromatography system was used for separation and identification of OTC antibiotic. High performance liquid chromatographic (HPLC) method with ultraviolet detection for the analysis of OTC is applied to the determination of OTC purification. (Author). 24 refs.