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A novel process using O2-enriched air supply was used to suppress the C-terminal proteolytic degradation of recombinant hirudin (r-hirudin) from Saccharomyces cerevisiae. When dissolved O2 was controlled above 20% saturation level using normal air, inactive forms of C-terminally truncated hirudin were observed in culture broth from 48 h of fermentation. The use of O2-enriched air giving above 40% saturation of dissolved O2 suppressed the proteolytic degradation and hence the formation of truncated forms of inactive r-hirudin until 60 h of fermentation.