Five different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis(trifluoromethyl)sulfonyllimide anions, were used to investigate the scope and limitations of these new solvents as media for penicillin G acylase-catalyzed reactions. Deactivation of the native enzyme in ionic liquids (ILs) and in organic solvents (toluene, dichloromethane and 2-propanol) at low water content and 40 degrees C was investigated using the hydrolysis of penicillin G as activity test. Native penicillin G acylase shows greater stability in IL media than in organic solvents. For example, a half-life time of 23 h was obtained in 1-ethyl-3-methylimidazolium bis{(trifluoromethyl)sulfonyllimide, [emim(+)][Tf2N-], which was about 2000-fold higher than that in 2-propanol. An enhancement of the PGA stability was observed by the presence of substrate in ionic liquids based on tetrafluoroborate and hexafluorophosphate anions, achieving the highest increase of the half-life time in 1-butyl-3-methylimidazolium hexafluorophosphate ([bmim(+)][PF6-]), which was about 9-fold higher than the half-life time in the absence of substrate.