Carboxypeptidase Y is widely used in peptide sequencing and mass spectrometry. PRC1 coding for proteinase C from Saccharomyces cerevisiae was expressed in Pichia pastoris GS115 as procarboxypeptidase Y with a yield of similar to 605 mg/l in shake-flasks after 168 h induction with 1 % (v/v) methanol. This precursor of carboxypeptidase Y was cleaved by endogenous proteinases of P. pastoris and released into the fermentation broth as active carboxypeptidase Y within 2 weeks at 10 A degrees C, which facilitated the preparation of mature carboxypeptidase Y. The recombinant enzyme was purified. It was optimally active at 30 A degrees C and pH 6.0, with an optimal activity of similar to 305 U/mg using benzyloxycarbonyl-l-phenylalanyl-l-leucine as substrate. This is the first report about high-level expression and activation of carboxypeptidase Y in P. pastoris.