AbstractTylosin‐producing Streptomyces fradiae was cultured on a synthetic medium with a high glutamate–glucose ratio. Tylosin batch fermentations with this medium were characterized by a high initial specific production rate of tylosin (qtylosin, mg/g h) that decreased as the fermentation progressed. Continuous feeding of glutamate, glucose, and methyloleate at a constant feed rate initiated during the period of high qtylosin had been shown to produce some increase in tylosin productivity. By using a cyclic feeding strategy, it was possible to increase tylosin productivity further. Tylosin fed‐batch fermentations with glutamate and glucose being fed to the culture in cyclic square‐wave profiles with methyloleate in excess showed several‐fold increase in final qtylosin and tylosin titers. By varying cycle amplitudes and period of the substrates, it was found that maximum tylosin productivity occurred when the glutamate cycle amplitude was 600 mg/L and that of glucose was 42.5 mg/L per cycle period of 24 h. With these cycle amplitudes of glutamate and glucose, the tylosin cyclic fed‐batch culture also showed high cellular uptake of methyloleate. Decreasing or increasing glucose cycle amplitude at fixed glutamate amplitude lowered tylosin production, and no further stimulation of tylosin synthesis was observed when α‐ketoglutarate was supplemented to the cyclic substrate feeds. Under optimum cyclic conditions it was possible to maintain linear tylosin accretion and a constant value of qtylosin up to 240 h.