The rate of substrate (tylosin) supply, pH, dissolved oxygen concentration, and temperature for bioconversion of tylosin to acetyl-isovaleryl tylosin (AIV), were optimized in a flask and a 3-l jar fermenter. An AIV concentration 9.3 g/l was achieved when the pH was controlled at 7.0 and the tylosin supply rate was maintained at 200 mg/l/h during the period of bioconversion of tylosin to AIV after the cell growth phase. Based on the data obtained for the Bask and jar-fermenter cultures, the bioconversion process was successfully scaled up to a 200-l fermenter. In this pilot-scale fermenter, 13.8 g/l of AIV was obtained at a yield of 83% based on the amount of tylosin supplied.