Decolourisation of the recalcitrant dye Remazol Brilliant Blue R (RBBR) by the fungus basidiomycete Pleurotus ostreatus was investigated. P. ostreatus is able to decolourise RBBR on agar plate. When grow in liquid media supplemented with veratryl alcohol, the fungus completely decolourises RBBR in 3 days. In these conditions, P ostreatus produces among other enzymes, laccases, veratryl alcohol oxidase and dye-decolourising peroxidase but only laccases seem to be responsible of RBBR transformation. Two purified laccases (POXC and POXA3) were found able to degrade RBBR in vitro, in the absence of any redox mediators. These laccases differ significantly in their efficiency of decolourisation of the tested dye, as suggested by comparison of their catalytic efficiency (k(cat)/K-m values) towards RBBR. Furthermore, using a mixture of both POXC and POXA3 a remarkable improvement in the reaction rate and in the final level of dye decolourisation was observed. The extent of RBBR decolourisation by laccase mixture also depends on incubation temperature and enzyme concentration. The dye is decolorised by laccase isoenzymes most efficiently under acidic conditions. Treatment of RBBR with the laccase mixture reduced its toxicity by 95%. (C) 2004 Elsevier Inc. All rights reserved.