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Nitrogen regulation in tylosin production by Streptomyces fradiae NRRL 2702 was studied in chemostat culture using a soluble synthetic medium. The maximum value of specific tylosin formation rate (qTYL) was 1.13 mg g−1 h−1 at the specific growth rate (μ) of 0.05 h−1, andqTYL decreased with increasing levels of the specific growth rate after reaching a rate of 0.1 h−1. The optimum conditions for tylosin formation were that the specific ammonium ion uptake rate (qN) and μ were 0.13 mmol g−1 h−1 and 0.05 h−1, respectively. The specific formation rates of threonine dehydratase (TDT) and tylosin were repressed by high levels of specific ammonium ion uptake rate. This study showed the adaptation to chemostat cultures of the nitrogen regulation of tylosin fermentations.