High concentration production of an antibiotic, thiostrepton, was achieved by the fedbatch culture of Streptomyces laurentii. To produce thiostrepton efficiently, the pH of the medium had to be maintained in a very narrow range between 6.0 and 6.2. As the substrates, not only glucose but also natural nutrients such as defatted soy bean meal and corn steep liquor were demanded. All of these substrates (multi-substrate) had to be supplied during the cultivation, pH was used as the indicator to detect the deficiency of substrates. When the glucose in the medium had been exhaustively consumed, the pH increased immediately; the multi-substrate solution (MS-solution) was then supplied. The composition of the feeding solution was determined from data obtained in batch cultures. During the fed-batch culture, glucose concentration was kept lower than 5 g/l by regulating the feed amount of MS-solution with this pH-stat modal control. By this control strategy a high concentration of thiostrepton, 10.5 g/l, was obtained, while total cell mass concentration reached 157 g/1. The productivity of thiostrepton was greatly increased compared with the conventional batch culture.