The C-terminal region of alpha-amylase (Amy1A/3D) secreted from recombinant Saccharomyces cerevisiae and Pichia pastoris was characterized by use of an anti-peptide antibody against this region of Amy1A/3D. In flask-culture of S. cerevisiae cells, the activity of a-amylase increased processing of the C-terminal region, a serine- and eysteine-protease inhibitor leupeptin was added to culture broth time to time. When leupeptin was adequately added, the activity of alpha-amylase with the intact C-terminus was 7 times the activity obtained without addition of leupeptin. Although in flask-culture of P. pastoris cells, the activity of alpha-amylase and cell density were much higher than those of S. cerevisiae, the protease activity was also much higher than that in S. cerevisiae, and thus the percentage of alpha-amylase with the intact C-terminus became about 60% in secreted alpha-amylase after 72 h induction. On the other hand, in cultivation of P. pastoris cells with a jar fermentor under controlled DO and the methanol concentration, the percentage of alpha-amylase with the intact C-terminus remained about 90%. Therefore, it is important to optimize fermentation conditions for depressing secretion of protease in production of recombinant proteins by secretion.