Water-in-hexane macro- and micro-emulsions stabilized by sodium bis-(2-ethylhexyl) sulfosuccinate (AOT) were used for selective permeabilization of Escherichia coli cells to extract penicillin acylase (3.5.1.11). Various organic solvents and surfactants were compared for the yield and purification of the enzyme. Recoveries upto 144% with respect to sonication and specific activity of the enzyme upto 26 units mg(-1) have been achieved. Due to low solubilities of AOT and aliphatic hydrocarbons in water the recovered enzyme was free from contaminants. A possible mechanism for cell permeabilization and enzyme purification by reverse micellar treatment is proposed.