Reversed micelles including the molecular chaperone GroEL were applied to the protein refolding of denatured RNase A. The molecular chaperone was successfully functionalized in the water pools of the reversed micelles sharing a structural size of 15-25 nm. The refolding yield of RNase A in the reversed-micelle/GroEL system was much greater than that of spontaneous renaturation. The refolding yield mediated by GroEL in the reversed micelles was strongly dependent on the presence of ATP or Mg2+, suggesting that the GroEL hosted in the reversed micelles was biologically active in the micelles. Under the optimum conditions, this novel refolding technique could completely renature the denatured RNase A in 1 h.