To achieve overproduction of the thermophilic protease aqualysin I (AQI), batch and fed-batch cultures of Escherichia coli TG1 transformed with an expression plasmid pAQN were investigated. The AQI gene, derived from Thermus aquaticus YT-1, was inserted under the control of the tac promoter in pAQN. During the fed-batch culture of recombinant E. coli TG1, acetic acid accumulated to a concentration of above 10 g/l in the culture medium. This accumulation was closely related to the glucose concentration of the culture medium, and it affected not only cellular growth but also the rate of AQI production. When 12 g/l of acetic acid was added to a batch culture at the time of IPTG induction, the rate dropped to 63% of the maximum value for batch culture without the addition of acetic acid. To suppress acetic acid accumulation, fed-batch cultures were carried out using an on-line glucose analyzer and HPLC unit to control the glucose feed rate, while the acetic acid concentration was monitored in real time. When the DO and glucose concentrations were maintained at 50% saturation and less than 0.3 g/l, respectively, the acetic acid concentration remained at a low level. Under these culture conditions, 18 g/l of cells and 33 kU/ml of enzymatically active AQI were obtained.