Hemocyanin and phenoloxidase are both copper-binding proteins involved in the immune system for a wide range of animal species. In crayfish, these proteins were purified and characterized from plasma and hemocytes, respectively. Recently, we have reported that the processing of one of the hemocyanin subunits occurs by a proteolytic cleavage under acidic conditions which results in the release of an antibacterial peptide designated as astacidin 1 from the C-terminus [J. Biol. Chem. 278 (2003) 7927]. In the present paper, we show that cleavage of crayfish hemocyanin subunit 2 at the N-terminal part results in that the processed hemocyanin exhibits phenoloxidase activity. The calculated mass of the cloned hemocyanin 2 is 78,372 Da, which corresponds to the size obtained after SDS-PAGE under reducing conditions of the purified hemocyanin and pI is estimated to be 5.70. The complete hemocyanin 2 sequence shows 74% and 44% similarity with hemocyanin 1 and prophenoloxidase of crayfish, respectively. Crayfish hemocyanin exhibited phenoloxidase activity in presence of trypsin, but no activity could be detected if treated with sodium dodecyl sulfate. These results show that hemocyanin of crayfish is involved in several immune responses such as an oxygen carrier protein, as a precursor for an antibacterial peptide, and a molecule with phenoloxidase function. (C) 2004 Elsevier Inc. All rights reserved.