Recently, ionic liquids (ILs) have received increasing attention as attractive solvents in chemical and biotechnological applications. The different influences of ionic liquids and their associated ions indicate the cooperative functioning of both cation and anion of ILs in affecting the enzyme performance. A comparative study on the influence of 1-butyl-3-methylimidazolium (BMIM Cl and BMIM BF4) and 1-methylimidazolium (HMIM Cl and HMIM BF4) on the kinetic parameters of the thermophilic alcohol dehydrogenase from Thermoaerobacter brokii (TBADH) and the mesophilic enzyme from yeast (YADH) was carried out. Values of 26 mM and 1250 mol min -1 mg were calculated for YADH K m and V max , respectively, using ethanol as a substrate. For TBADH the K m for 2-propanol was 7 mM and the V max was 45 mol min -1 mg. As the concentration of ionic liquids increased, K m increased and V max decreased gradually. The inhibition profile indicated mixed inhibition and the K i (affinity of IL for the enzyme) and K i (affinity of IL for enzyme-substrate complex) values were calculated. The thermophilic enzyme activity in the presence of 0.25% (v/v) HMIM Cl and HMIM BF4 reduced to 17% of the initial reaction rate whereas the mesophilic alcohol dehydrogenase (YADH) in the presence of 0.25% (v/v) HMIM Cl and HMIM BF4 demonstrated 95% of the initial activity indicating that the thermophilic alcohol dehydrogenase is more sensitive to HMIM. The remaining activity of 46% was obtained for TBADH in 4% (v/v) BMIM Cl and BMIM BF4 while the YADH exhibited 53% and 9% of the maximum activity in BMIM Cl and BMIM BF4 respectively. The associated conformational changes of the secondary and tertiary structures of the enzymes caused by ILs were examined by circular dichorism (CD) and fluorescence techniques. The effect of ILs on enzyme performance can be related to their ionic nature via interactions with the enzyme structure, the substrate and water molecules associated with the enzyme.