The kinetics of hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by alpha-chymotrypsin (alpha-CT), in reverse micellar solutions formed by glycerol (GY)-water (38% v/v) mixture/sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/n-heptane has been determined by spectroscopic measurements. To compare the efficiency of this reaction with that observed in micelles with water in the core, as well as in the corresponding homogeneous media, the reaction was also studied in water/AOT/n-heptane reverse micellar solutions and in both homogeneous media (water and GY-water, 38% v/v mixture). In every media, alpha-CT was characterized by the absorption and emission spectra, the fluorescence lifetimes, and the fluorescence anisotropy of its tryptophan residues. The effect of AOT concentration on the kinetic parameters obtained in the micellar systems was determined, at a constant molar ratio of the inner polar solvent and surfactant. Moreover, the data obtained allowed the evaluation of the 2-NA partition constant between the organic and the micellar pseudophase. It is shown that the addition of GY to the micelle interior results in an increase in the catalytic properties of alpha-CT. The fluorescence anisotropy studies in the different media show that the addition of GY increases the viscosity as compared with the aqueous systems. It seems that the GY addition to the reverse micellar aggregates results in a decrease of the conformational mobility of alpha-CT, which leads to an increase of the enzyme stability and activity.