The effect of chain length on the enzymatic hydrolysis of various p-nitrophenyl esters was investigated. Specifically, the hydrolysis of various esters p-nitrophenyl butyrate (PNPB), p-nitrophenyl caprylate (PNPC), p-nitrophenyl laurate (PNPL), p-nitrophenyl myristate (PNPM) and p-nitrophenyl palmitate (PNPP) was studied in supercritical carbon dioxide (ScCO2) with lipase (Novozym 435). This indicates that the conversion of nitrophenyl esters decreases with increasing chain length. The effect of various parameters such as amount of water added, temperature, and enzyme loading was studied. The optimum temperature for the hydrolysis of PNPB and PNPC was 50°C but was 55°C for PNPL, PNPM, and PNPP in ScCO2. The reactions were also conducted in acetonitrile as the solvent, and it was found that the reactions reach equilibrium much faster in ScCO2 than in acetonitrile. The kinetics of the hydrolysis reactions were modeled using a Ping Pong Bi Bi model. © 2007 Humana Press Inc.