The catalytic activity of α-chymotrypsin (α-CT) for the hydrolysis of p-nitrophenyl acetate has been studied in oil-in-water microemulsions formed by cetyltrimethylammonium bromide, n-butanol and alkanes (organic solvents). The specificity of α-chymotrypsin regarding the chain length of the organic solvent (n-hexane, n-heptane and isooctane) in CTAB/n-butanol microemulsions was tested in hydrolytic reactions of PNPA. The stability and activity of α-CT depend upon organic solvents. Under all the conditions employed, the reaction follows a Michaelis–Menten mechanism. The second order rate constant, k 2 was determined for α-CT catalyzed hydrolysis of p-nitrophenyl acetate at 27°C. The chain length of oil influences the k cat and K M . The CTAB/n-butanol/isooctane (z=1.07) system of o/w microemulsion shows the higher activity and stability of α-CT.