Lipases from Rhizomucor Miehei, Candida antarctica, and Fusarium solani pisi were chemically modified with the aim to improve their catalytic properties in organic solvents. The chemical modifiers. two activated polyethylene glycol derivatives and activated n-octanol, were covalently linked to lysine residues at the surface of the enzyme leading to varying surface hydrophobicities. The modified lipases were tested for hydrolytic activity in water and for transesterification activity in the organic solvents o-xylene, tert-butyl methyl ether, tert-butanol, and 2-butanone. Whereas the hydrolytic activity was only slightly affected by the modifications, the transesterification activities were influenced strongly even though the modified lipases were still not soluble in organic solvents. The most effective modifier is tryesyl-activated polyethylene glycol 2000 monomethyl ether, activating lipases up to 27-fold in organic solvents while it is the least hydrophobic. The more hydrophobic modifiers, tresyl-activated polyethylene glycol 400 mono-octyl ethyl (tOPEG) and tresyl-activated octanol (tOCT), may lead to inactivation. Co-lyophilization of unmodified Candida antartica lipase B (CALB) with additives such as polyethylene glycol dimethyl ether and crown ether also positively affects the activity of CALB in organic solvents. However, we found that covalent linking of MPEG to CALB is more effective because the activation by additives is partially lost during washing of the enzyme for reuse. The thermostability of CALB in o-xylene is not affected by modification, whereas in 2-butanone the thermostability is decreased by MPEG modification and increased by OPEG or OCT modification. Our results suggest that MPEG positively influences the porosity of the lipase aggregates in organic media, whereas OPEG and OCT induce tighter aggregates.