This study examined the effects of two methods of methanol feeding, DO-stat and methanol concentration control, in fed-batch and continuous cultures of Pichia pastoris on cell growth and single-chain variable fragment antibody (scFv) expression. By maintaining the methanol concentration at 3.9 g l(-1) in fed-batch culture, a scFv concentration of 198 mg l(-1) was obtained. In continuous culture using both methanol feeding methods, the scFv concentration in the fermentation broth increased with a decreasing dilution rate. A maximum scFv concentration of 810 mg l(-1) at a dilution rate of 0.0094 h(-1) was obtained by maintaining the methanol concentration at 3.9 g l(-1). Although the specific methanol consumption rate was the same for both methods, the specific productivity of scFv was higher in methanol concentration control from 0.0094 to 0.049 h(-1) than it was in DO-stat control. Therefore, continuous culture with methanol feeding by the concentration control method shows promise for the industrial scale production of recombinant proteins by Pichia pastoris.