Sample preparation methods based on the use of proteolytic and cell wall digesting enzymes for the speciation analysis of selenized mushroom were investigated. The sample (Agaricus bisporus;160 mug total Se per g sample) was grown on compost supplemented with selenized yeast. Experiments were carried out to elucidate the possible role of the cell wall digesting enzymes Lysing enzyme and Driselase - in the improvement of extraction efficiency with and without inhibiting proteolysis during cell wall digestion. A 3-step procedure applying Lysing enzyme and pronase gave the highest extraction efficiency (89%); however, the best species recovery was achieved by a one-step proteolytic procedure. All the procedures of selenium speciation were controlled by independent ICP-AES analysis measuring the total amount of selenium.