The ability of Trametes villosa laccase, in conjuction with 1-hydroxybenzotriazole (HBT) as mediator and alkaline extraction, to remove lignin was demonstrated during treatment of wood (Eucalyptus globulus) and non-wood (Pennisetum purpureum) feedstocks. At 50 Ug(-1) laccase and 2.5% HBT concentration, 48% and 32% of the Eucalyptus and Pennisetum lignin were removed, respectively. Two-dimensional nuclear magnetic resonance of the feedstocks, swollen in dimethylsulfoxide-d(6), revealed the removal of p-hydroxyphenyl, guaiacyl and syringyl lignin units and aliphatic (mainly β-O-4'-linked) side-chains of lignin, and a moderate removal of p-coumaric acid (present in Pennisetum) without a substantial change in polysaccharide cross-signals. The enzymatic pretreatment (at 25 Ug(-1)) of Eucalyptus and Pennisetum feedstocks increased the glucose (by 61% and 12% in 72 h) and ethanol (by 4 and 2 g L(-1) in 17 h) yields from both lignocellulosic materials, respectively, as compared to those without enzyme treatment.