The activity of several commercial and genetically engineered lipases, already immobilized on octyl-Sepharose, in aqueous solutions containing ionic liquids (ILs) in a molar concentration of 0.01 mol L-1 was investigated. Strong variations in the hydrolytic activity values were found depending on the lipase, the ionic liquid and the substrate used. The most interesting results were found in the engineered variants of Geobacillus thermocatenolatus lipase (GTL). The activity of GTLσ-L230C variant in the monodeacetylation of per-acetylated glucal increased seven fold when using 1-ethyl-3-methylimidazoliumhexafluorophosphate [emim[PF6] and fivefold when using 1-ethyl-3-methylimidazolium methyl sulphate [emim][MeSO3]. In general, ILs containing BF4- had a very negative effect on the enzyme activity. Moreover, improvements in the enantio and regioselectivity of the immobilized lipases were obtained by adding ILs to the reaction medium. The regioselectivity of Candida rugosa lipase in the hydrolysis of peracetylated thymidine improved (from 72% to 81% yield of C-5 monodeprotected product) in the presence of [emim][PF6]. More interestingly, the addition of 1-butyl-2,3-dimethylimidazolium hexafluorophosphate [bdmim][PF6] improved the regioselectivity of immobilized GTLσ-L230C variant in the hydrolysis of peracetylated glucal, from 78% to 96% yield of the C-3 monodeprotected product. Circular dichroism and fluorescence experiments revealed conformational changes in the tertiary structure of the enzyme induced by the ionic liquid which could explain these phenomena.