Proteins were extracted and highly concentrated by their partitioning to a bicontinuous, middle phase of W_III microemulsion system. The binary surfactant system consisted of Aerosol-OT, or AOT, and a cleavable, 1,3-dioxolane surfactant possessing poly (ethylene glycol) monomethyl ether, or MPEG as its hydrophile. A binary system based on a 1,3-dioxolane surfactant possessing an average degree of polymerization for MPEG 7.2 was successful for extracting lysozyme and bovine serum albumin from 1 g/L aqueous solutions, due to the attractive driving forces imposed by AOT, but required moderately high temperatures (~40°C) for the formation of suitable 3-phase microemulsion systems. Use of binary 1,3-dioxolane surfactant mixtures with MPEG degree of polymerization 7.2 and 3.0, in addition to AOT, resulted in 3-phase microemulsion systems at 25°C. At least 90% forward extraction of several different proteins (lysosyme, a-chymotripsin, and cytochrome C) to the middle, bicontinuous, microemulsion phase occurred when an electrostatic driving force existed between the protein and AOT. In contrast, only 5% of the protein pepsin was extracted using pH7 buffer, due to the protein's net negative charge. The experiments were done at different concentrations and proportions of surfactant and protein. Back extraction was achieved by changing the water solution of the 3-phase system with low pH solution or high salt concentration.