Abstract The influences of the thermodynamic water activity ( a w) and the reaction medium hydrophobicity on the synthesis of ascorbyl palmitate, in the 2-methyl-2-butanol as a solvent, in the presence of the immobilized Candida antarctica lipase as a biocatalyst, were investigated. The effect of moisture on the equilibrium and the rate of the transesterification reaction, involving palmitic acid methyl ester and ascorbic acid, was evaluated through measurements made in reaction media, which were initially pre-equilibrated in controlled water activity atmosphere. The catalytic activity of the enzyme as well as the production were significantly affected by the pre-fixed water activity level, best results being obtained for the lowest a w values tested. An increase in the acyl donor/ascorbic acid molar ratio ( R) up to 9 was demonstrated to promote the activation of the lipase, leading to an ascorbyl palmitate production of 19 g l −1 after a reaction time of 5 h. That performance was not reached in a reaction medium of equal log P level, where methyl palmitate was partly substituted by a hydrophobic solvent. Hydrophobic property of the bulk organic phase interacting with the catalyst was suggested to be a necessary but insufficient factor promoting lipase activation.