The recovery and recycling of cellulases during fed-batch hydrolysis of cellulosic substrates derived from SO2-impregnated steam-treated Eucalyptus viminalis chips was examined. An initial enzyme loading of 10 FPU and 25 CBU g(-1) cellulose was shown to be effectively recovered and recycled to hydrolyse seven consecutive batches of pretreated substrate at a 5% (w/w) cellulose concentration. Although the alkali-treated residue derived from steam-treated eucalyptus did not provide good cellulase recovery, subsequent alkaline peroxide treatment of this residue dramatically enhanced cellulase recycling efficiency. A glucose production rate of 26.1 +/- 1.7 mg mL(-1) d(-1) was obtained for seven consecutive days, with an elapsed time of 24 h between each recycling step, without any requirement for further addition of cellulases after the first hydrolysis step. However, a considerable loss of both protein and enzymatic activity was observed throughout the experiment. In preliminary work, an excess of cellobiase activity was added at the beginning of every new hydrolysis step to avoid cellobiose accumulation in the reaction mixture. This was shown to be unnecessary when an additional step of ultrafiltration was used to recover and recycle the cellobiase activity present in the hydrolysates.