The influence of three system parameters [bis(2-ethylhexyl)sulfosuccinate (AOT) concentration, pH, and temperature] on the selective separation of cytochrome c, lysozyme, and ribonuclease A from buffer solution and a filtered fermentation broth was examined. It was found that a minimal AOT concentration exists for greater than or equal to 90% extraction from a buffer solution, and this concentration depends on the pH of solution and hydrophobicity of the protein. Extraction from filtered broth resulted in a reduction of the minimal concentration for both cytochrome c and lysozyme, while for ribonuclease A it was unchanged. It appears that certain broth constituents act as cosurfactants and reduce the charge repulsion between the surfactant headgroups leading to smaller micelles. This is turn led to a reduction in the pH range, although extraction yields were unaltered. The water content (W-0) was measured in a reverse micellar phase in contact with buffer, filtered fermentation broth, and molecular weight fractionated or extracted broth. This data led to the conclusion that the broth constituents responsible for changes in the minimal AOT concentration and W-0 were low molecular weight positively charged amino acids and phospholipids. Finally, response surface methodology was used to optimize key system parameters in order to maximize protein extraction, and this technique minimizes the experiments required. By using these optimized conditions, a mixture of the three proteins could be recovered from a filtered broth with high yields (70-97%) and high purity. Furthermore, the initial protein concentration was found not to influence protein recovery.