Two strictly related laccase isoenzymes (POXA3a and POXA3b), produced by Pleurotus ostreatus in copper supplemented cultures, have been purified and characterised. Both the native proteins were found to be constituted by a large subunit (67 kDa) and a small subunit (18 or 16 kDa). Peptide mapping of the 18 and 16 kDa polypeptides from POXA3a and POXA3b suggests the identity of the 18 kDa subunits and the generation of the 16 kDa polypeptides from the 18 kDa ones. Structural data on POXA3a and POXA3b do not allow ascertaining significant differences between the two isoenzymes. On the other hand, dissociation of POXA3a complex is observed in 3 M urea, whilst POXA3b complex is not dissociated even in 6 M urea. Evidences are reported on the role played by extracellular proteases in the activation of these isoenzymes. The sequence of a unique gene and of the corresponding cDNA, encoding the 67 kDa POXA3 subunit, has been determined. (C) 2003 Elsevier Science Inc. All rights reserved.