The optimal culture conditions for attaining high levels of expression of mouse α-amylase in the methylotrophic yeast Trichoderma reesei were investigated. Cell growth and enzyme production were found to be optimal at a temperature of 28 °C and a pH of 6.0. Yeast extract and peptone were the most appropriate nitrogen sources for the expression of α-amylase. When a mixture of 25 g/l peptone and 20 g/l yeast extract was used as the nitrogen source, α-amylase activity reached 300 U/ml. The expression of α-amylase in a fed-batch culture using methanol as the sole carbon source reached 810 U/ml at 4 days of culture. However, when a methanol-to-glycerol mixture was used at ratio of 1:0.5 at a feed rate of 3 g/h, the maximum α-amylase activity was 2.23 kU/ml. Under the optimal culture conditions, when 40 g/l of a mixture of yeast extract and peptone (1:1) was added to the culture at 3 and 5 days the final α-amylase activity increased to 3.3 kU/ml, which was 4.5- and 2.5-fold higher than the activity in the batch culture and in the fed-batch culture with feeding a mixture of methanol and glycerol at a ratio of 1:0.5, respectively. This study demonstrates that an addition of the appropriate amount of an organic nitrogen source to the fed-batch culture is effective to improve high level of expression of mouse α-amylase in Trichoderma reesei.