Candida rugosa lipase (CRL) was used to catalyze the regioselective acylation of methyl 6-O-trityl beta-D-glucopyranoside in supercritical CO2 (SCCO2), using vinyl acetate as the acyl donor and obtained final conversion values up to 91.4%. The effect of pressure, temperature, enzyme quantity and sugar/ester molar ratio on the catalytic efficiency and regioselectivity of CRL was evaluated. The regioselectivity of the reaction was shifted towards the synthesis of 3-O-acetyl 6-O-trityl beta-D-glucopyranoside and this is a function of the chemico-physical parameters of the supercritical medium employed. In fact, by varying the CO2 pressure used in the process up to 20 MPa at constant temperature, we were able to obtain the formation of the sole 3-O-acetyl 6-O-trityl beta-D-glucopyranoside regioisomer. A kinetic study was also performed and the relative kinetic parameters calculated. (C) 2007 Elsevier B.V. All rights reserved.