A highly-active tyrosinase (H subunit) isoform has been purified from a commercial crude extract of Agaricus bisporus by a specific, two step-hydrophobic chromatography cascade process based on the differential adsorption of the proteins from the extract to hydrophobic-functionalized supports. At first, commercial, crude tyrosinase from Agaricus bisporus (AbTyr) dissolved in aqueous media was added to octadecyl-Sepabeads matrix at 25 degrees C. Under these conditions, the support specifically adsorbed a protein with a molecular weight of 47 kDa which showed no tyrosinase activity. The known H subunit of tyrosinase from Agaricus bisporus (45 kDa, H-AbTyr) and another protein of 50 kDa were present in the supernatant. Sodium phosphate buffer was added to adjust the ionic strength of the solution up to 100 mM and Triton X-100 was added (final concentration of 0.07% v/v) to control the hydrophobicity effect for both proteins. This solution was offered again to fresh octadecyl-Sepabeads support, immobilizing selectively the H-AbTyr and leaving exclusively the 50 kDa protein as a pure sample in the supernatant. This tyrosinase isoform of 50 kDa was almost 4-fold more active than the known H-TyrAb, with a specific tyrosinase activity of more than 38,000 U/mg.