wIn recombinant Pichia pastoris fermentation for hirudin production in a 5 l fermenter, a new strategy was explored to match the short fermentation time at low NH4+ 4 concentration with decreased hirudin degradation at high NH4+ concentration. A combination of a defined medium containing initial 0.025 M NH4+ with NH4+ addition up to 0.6 M in the growth phase was achieved in both the improvement of hirudin production and the repression of hirudin degradation. Intact and total hirudin reached 2.63 g l(-1) and 4.25 g l(-1), respectively.