3-phase microemulsion systems consisting of mixed Aerosol-OT (AOT) / pH-degradable 1,3-dioxolane alkyl ethoxylate nonionic surfactant (CK) have been employed to isolate proteins. Equal volumes of aqueous-containing protein solution and surfactant-containing organic solvent are gently mixed; a 3-phase system consisting of "excess" wate and oil phases and a surfactant- and protein-rich bicontinuous microemulsion middle phase results. Appropriate microemulsion systems can be prepared by proper adjustment of the relative amounts of AOT and CK, the head group size distribution for CK, and the ionic strength, with the system formed at its hydrophilic-lipophilic balance temperature to facilitate rapid phase separation. Proteins have been extracted into the middle phase of these systems at yields of >90%, driven by electrostatic or hydrophobic attractive forces between the enzyme surface and AOT, increasing the concentration of protein 5-fold. The middle phase reaches saturation of protein when the initial aqueous phase contains 2-5 mg/mL, depending on the protein type. The degree of success for forward protein extration and recovery of microemulsion-encapsulated proteins ("back-extraction"), typically ~70-90%, depends on the hydrophobicity of the protein and the medium's composition. Several schemes for back-extraction have been explored and will be compared, with the best results obtained using brine as a stripping solution, and adding a small amount of nonionic surfactant. Recovered proteins possess 100% of their initial specific activity.