Annexin A2, a Ca2+-dependent phospholipid binding protein, is abundantly expressed in various human organs, which exists as either a membrane-associated, cytosolic or soluble form in serum. We constructed expression systems for recombinant human annexin A2 (rhA2) using Pichia pastoris. The systems are designed to secrete rhA2 as either the N- or C-terminally His(6)-tagged form to facilitate purification. Both types of rhA2 were overexpressed, but in the N-terminal-truncated form as revealed from the results of N-terminal amino acid sequencing and Western blotting. Therefore, further purification of N-terminally His,-tagged rhA2 was not feasible because of the removal of the N-terminal His(6)-tag sequence. C-terminally His(6)-tagged rhA2 was expressed as either a glycosylated or a nonglycosylated form, and the nonglycosylated form was purified using the combination of nickel-immobilized affinity, concanavalin A and cation exchanged column chromatographies. The solid-phase binding of rhA2 was examined by enzyme-linked immunosorbent assay (ELISA), which revealed the specific reactivity of rhA2 against an anti-annexin A2 monoclonal antibody. These results suggest that the expression system using P pastoris is useful for the preparation of rhA2 that is applicable to the ELISA detection of the anti-annexin A2 antibody.