Softwood substrates are recalcitrant in enzymatic hydrolysis to reducing sugars, in part because of unproductive binding of enzymes on lignin. Interactions between polyethylene glycol (PEG) and a softwood substrate were characterised in order to estimate the PEG loadings required for effective blocking of unproductive enzyme binding. For the first time, the adsorption of Rhodamine labelled PEG (M-w 3400) on thermo-mechanically treated Pinus radiata fibres was characterised by fluorimetry, giving a best-fit binding constant of 68 L g(-1). This was considerably stronger than published binding constants of 6 L g(-1) for enzymes on lignin, accounting for the success of PEG as an additive. Glucose yields from enzymatic hydrolysis of the same substrate, when unlabelled PEG (M-w 4000) was used, were consistent with a PEG adsorption capacity of 0.01-0.02 g g(-1) substrate. Thus, PEG was shown to be effective at affordably low loadings and further mathematical modelling work predicts savings correspond to 6 kg PEG per tonne when pretreated Pinus radiata substrate was processed at higher substrate concentration. (C) 2014 Elsevier Ltd. All rights reserved.