The recovery of four proteins by means of the classical reverse micellar two-phase system consisting of brine, AOT (Aerosol OT, sodium di-2-ethyl-1-hexyl sulfosuccinate), and iso-octane has been studied. A complete cycle of extn.-forward and backward- was performed each time to optimize the AOT concn. with respect to the overall protein yield. These expts. have shown that max. global yields can be obtained at quite low surfactant concns. in the range of 2-20 mM AOT. These low surfactant concns. were beneficial for the operation of the extn. process owing to facile phase sepn. In addn., the partitioning of surfactants in the reverse micellar two-phase system, as well as the assocn. of surfactants with proteins in the aq. phase, have been studied because they are of primary importance for assessing the feasibility of reverse micellar protein extn. The question of the fate of the surfactant has been addressed by using radioactively labeled AOT. By varying the initial AOT concn. in the nonpolar phase, optimum surfactant concns. were obtained with respect to minimal AOT contamination of proteins after a complete cycle of extn. The partitioning of AOT was studied in the absence, as well as in the presence, of proteins. Lowering the salt concn. favored the partitioning of AOT in the aq. phase. Therefore, most of the surfactant losses occurred during forward extn. A simple dialysis was effective for eliminating AOT. [on SciFinder (R)]