CD25 is a marker molecule expressed on many T and B cell neoplasms and on activated T cells that mediate organ transplant rejection and many autoimmune diseases. Single-chain variable fragments (scFvs) have many advantages over whole antibodies for use in antibody-targeted immunotherapy due to their small size. Daclizumab is an Food and Drug Administration-approved humanized anti-CD25 antibody. We attempted to produce a daclizumab-derived scFv, designated as Dmab(scFv), in Pichia pastoris. The Dmab(scFv) gene was designed based on the variable regions of the heavy and light chains of daclizumab, cloned into the yeast vector pPIC9K, and expressed in P. pastoris strain GS115. Under optimized conditions (culture medium pH, 6.0-6.5; methanol concentration added daily, 3.0%; inoculum density, OD600=60; induction time point, 72-96 h), the yield of soluble recombinant Dmab(scFv) was approximately 80 mg l(-1). Flow cytometry, immunohistochemistry, and Western blotting revealed that the purified Dmab(scFv) could bind strongly to the membrane of CD25-positive cells, including SNT-8 cells, ConA-stimulated human peripheral blood mononuclear cells (hPBMCs), and rat splenocytes, but not to CD25-negative cells, including Raji cells, unstimulated hPBMCs, and rat splenocytes. These results suggest that Dmab(scFv) produced in P. pastoris is active and specific toward CD25-positive cells and has potential for use in CD25-targeted immunotherapy.