The effect of dissolved oxygen (DO) tension on heterologous protein expression by the Pichia system was investigated by applying DO control bands of different set points and bandwidths in the DO-stat fed-batch fermentation. The model protein was elastase inhibiting peptide (EIP) that showed strong inhibitory activity against human neutrophil elastase with very high specificity. In the alcohol oxygenase I derepression phase, more frequent glycerol feeding by keeping the DO control band narrower resulted in a higher cell growth rate (0.10 h(-1)). In the methanol induction phase, maintaining a higher DO set point and allowing more frequent feeding significantly enhanced EIP expression. The EIP expression titre was increased about threefold to 846 mg l(-1) on average by raising the lower set point from 10 to 30% air-saturation. Both specific expression titre and methanol consumption rates were increased about threefold, which indicated that higher DO tension stimulated the methanol utilization pathway and improved the expression efficiency. To operate the DO-stats with higher DO set points, an oxygen enriching membrane system that could generate up to 38% oxygen partial pressure from the regular air was used as an alternative oxygen supplier to the traditional gas-mixing system consisting of an air compressor and a pure oxygen cylinder. (C) 2002 Elsevier Science Ltd. All rights reserved.