Solubilization of cytochrome (c) (cytc) in the water-in-oil microemulsion (ME) phase of bis(2-ethylhexyl) sulfosuccinate (AOT) was strongly time-dependent. An early quasi-equilibrium state was observed for systems produced by both phase transfer and injection. In the quasi-equilibrium state, large amounts of cytc and water were solubilized without precipitation. Spectroscopic measurements revealed that the conformation of cytc was denatured even in the quasi-equilibrium state; i.e., helix content decreased and anti (-\beta) structure formed in its place. This conformational change resulted in the formation of strong amphiphilic cytc, which was easily extracted in the ME phase and was localized in the interfacial zone of the ME droplets. The strong interaction of amphiphilic cytc with the ME interface changed the aggregate shape from spherical to cylindrical. With the lapse of time, further denaturation of cytc proceeded by adsorption of more AOT molecules, and some cytc molecules precipitated, being released from the ME droplets. The interaction between the cytc molecules and the ME aggregates in the equilibrium state was much weaker than in the quasi-equilibrium state, and the shape of ME aggregates with cytc became the same as that of those without cytc. The denatured conformation of cytc in the ME phase was restored to the native conformation by the back-extraction of cytc into (2.5 \mathrm{M} \mathrm{NaCl}) aqueous solutions. When cytc stayed in the ME phase over the quasi-equilibrium period, the fraction of back-extraction was lowered. It is important for the extraction of proteins such as cytc to complete the extraction operations, including the back-extraction process, within the quasi-equilibrium period. (1994 Academic Press, Inc.