A novel protein refolding method using reversed micelles has been developed, which could replace the conventional dilution method using a buffer solution. The novel refolding method enables efficient refolding at a high protein concentration. In the present study, denatured bovine heart cytochrome c was directly solubilized in AOT reversed micelles using the solid-liquid extraction technique. Results reveal that addition of urea in small amounts facilitates solubilization of denatured protein into the reversed micellar phase. Reversed micelles containing a high concentration of denatured cytochrome c could be easily prepared by the novel solubilization method. The nanostructural environment formed by the surfactant molecules in organic media Is considered to promote the renaturation of denatured proteins because the protein molecules are isolated from each other through the solubilization step. Although the recovery of entrapping proteins from the reversed micellar phase was known to be difficult in a conventional reversed micellar extraction operation, the addition of alcohol to the recovery phase improved the efficiency of back extraction. Therefore, we succeeded In recovering renatured cytochrome c from the reversed micelles. We demonstrated that the novel protein refolding method is very useful for the renaturation of denatured proteins.