Medium improvement was studied because chlortetracycline was not produced during fermentation by the medium composition which were commonly shown in many previous works. We found many factors which affect for the production of chlortetracycline by the modification of the medium. Among them, the concentration of divalent ions and the kind of carbohydrate were rather important. The concentration of divalent ions above the concentration included in the natural medium, shows negative effect on the production of chlortetracycline. In many previous works, sucrose was used as main carbon source on chlortetracycline fermentation, but it was found that starch was rather good for chlortetracycline fermentation. A spectrophotometric assay method involving the extraction of chlortetracycline with butanol(1) under controlled conditions has been developed. By use of this procedure, the chlortetracycline content of both fermented and pharmaceutical products may be determined. This method is applicable to quality control. In addition, the determination of cell growth was carried out by measuring the total hyphal length. This method showed rather good result for the determination of cell growth of Streptomyces sp. which grows well in the complexed medium where many insoluble solid contents are contained.