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White-rot basidiomycetes, the most common wood-rotting organisms, are characterized by their ability to produce extracellular oxidative enzymes, among which laccases are regarded as promising catalysts for many biotechnological applications. A significant obstacle to the exploitation of laccase-based bioprocesses is the large amounts of enzyme required. In this study the issue has been addressed by applying a classical breeding approach to increase laccase production yields in the white-rot fungus Pleurotus ostreatus. Starting from two different P. ostreatus varieties, three higher laccase-producing hybrids have been obtained by crossing selected compatible monokaryons. The three selected strains increased the titre of parental strains up to four fold, reaching an expression level of up to 100 000 U/L. One hybrid exhibited a more complex isoenzyme pattern, illustrating the potential of classical breeding to differentiate protein expression. [ABSTRACT FROM AUTHOR]