Experiments were carried out to investigate the effect of pretreatment by organic solvents with different hydrophobicities, functional groups and molecular constitutions as activation agents on initial esterification activity and secondary structure of immobilized Pseudomonas cepacia lipase. The results showed that esterification activity of immobilized P. cepacia lipase treated with organic solvents containing and -C N- functional groups was highest, followed by the one treated with -C-C- functional groups but the lowest with -OH and aromatics functional groups. An organic solvent with a branched structure was more favorable compared with a straight chain in terms of enhancing enzyme activity. Conformational studies via Fourier transform-infrared spectroscopy indicated that the catalytic activity variance was attributed to the secondary structure changes for immobilized P. cepacia lipase treated with organic solvents. Moreover, the effects of moisture, pH and temperature on the esterification activity of immobilized P. cepacia lipase were also addressed. (C) 2010 Elsevier Ltd. All rights reserved.