Solvent composition, system mobility and viscosity play a central role in the regulation of enzymatic activity. The aim of this study was to investigate the individual and combined effect of water activity (a(w)), bulk viscosity and glass transition temperature (T'(g)) on the activity of horseradish peroxidase (HRP) in buffered solutions. For this purpose, the water activity of the solutions was modulated using both a ionic (sodium chloride) and a non-ionic (sorbitol) ligand and viscosity changed upon the addition of maltodextrin. In viscous solutions characterized by different compositions, the effectiveness of a solute in the inhibition of the HRP activity was dependent both on the chemical properties of the solution, as described by a(w), and on the mobility of the system, as described by the inverse of viscosity and T - T'(g) Viscosity was the most important factor in the inhibition of HRP activity in solutions characterized by the same Tg value, but when T'(g) was changed, due to changes in the solutes composition, the latter became a key factor in the regulation of the enzyme activity. In salt-maltodextrin systems the water activity reduction limited HRP activity with higher efficiency at low viscosities whilst in sorbitol-maltodextrin systems, characterised by different T'(g) values, the a(w) lowering by sorbitol addition resulted in the increase of HRP activity depending on its effect on T'(g). (c) 2010 Elsevier Ltd. All rights reserved.