The white-rot fungus Trametes pubescens MB 89 is an excellent producer of the industrially important enzyme laccase. Extracellular laccase formation can be considerably stimulated by the addition of Cu(II) in the millimolar range to a simple, Calucose-based culture medium. When Using glucose, a typically repressing Substrate. as the main carbon source, significant laccase formation by T pubescens only started when glucose was completely consumed from the culture medium. In addition. the nitrogen source employed had an important effect on laccase synthesis. When using an optimized medium containing glucose (40 g 1(-1)), peptone from meat (10 g 1(-1)), and MgSO4.7H(2)O and stimulating enzyme formation by the addition of 2.0 mM Cu, maximal laccase activities obtained in a batch cultivation were approximately 330 U ml(-1). Under these conditions it was not necessary to add aromatic compounds that are routinely used as inducers of laccase in fungi. By applying a fed-batch strategy. in which a glucose solution was fed continuously to a cultivation of T pubescens so that the glucose concentration in the medium never exceeded a certain low, critical value, glucose repression could be avoided and production of laccase was almost doubled as compared to the batch cultivation (740 U ml(-1)).