The therapeutic enzyme asparaginase, which is used for the treatment of acute lymphoblastic leukaemia, is industrially produced by the bacteria Escherichia coli or Erwinia crysanthemi. In spite of its effectiveness as a therapeutic agent, the drug causes severe immunological reactions. As asparaginase is also produced by the yeast Saccharomyces cerevisiae, this microorganism could be considered for the production of the enzyme, providing an alternative antitumoral agent. In this study the ASP3 gene, that codes for the periplasmic, nitrogen regulated, asparaginase II from S. cerevisiae, was cloned and expressed in the methylotrophic yeast Pichia pastoris, under the control of the AOXI gene promoter. Similarly to S. cerevisiae the heterologous enzyme was addressed to the P. pastoris cell periplasmic space. Enzyme yield per dry cell mass reached 800 U g(-1), which was seven fold higher than that obtained using a nitrogen de-repressed ure2 da180 S. cerevisiae strain. High cell density cultures performed with P pastoris harbouring the ASP3 gene using a 21 instrumented bioreactor, where biomass concentration reached 107 g l(-1), resulted in a dramatic increase in volumetric yield (85,600 U l(-1)) and global volumetric productivity (1083 U l(-1) h(-1)). (c) 2006 Elsevier Inc. All rights reserved.