The interferons are cytokines with antiviral, antiproliferative and immunomodulatory activities. Among three major distinguished types, interferon-gamma (IFN-γ) is more popular for its specific properties in inhibition of cell growth and modulation of immune functions. Now the recombinant DNA techniques make it possible to express recombinant IFN-γ in E.coli in large amounts, however this may result in the formation of inclusion bodies, and the recovery of biologically active products becomes significant. As the minimal mechanism of GroEL-mediated protein folding, minichaperones, fragments encompassing the apical domain of GroEL, can facilitate the refolding of several proteins in vitro without requiring GroES, ATP, or the cage-like structure of multimeric GroEL. Here minichaperone (GroEL191～345)-mediated in vitro refolding of recombinant human interferon gamma (rhIFN-γ) in free and immobilized GroEL191～345 systems are studied. SDS-PAGE was performed with Laemmli's Tris-glycine buffer system for protein analysis. Protein concentrations were determined by using a modification of Lowrys method with bovine serum albumin as reference and total activities of renatured rhIFN-γ were measured by CPE method. The results showed that the presence of GroEL191～345 in the refolding buffer not only enhanced the specific activity, but also promoted the refolding efficiency. With the initial protein concentration of 100 mg/L, the protein yield and total activity of rhIFN-γ assisted by GroEL191～345 was 2.2 and 3 folds of that under spontaneous condition respectively. Optimal operating parameters in refolding of rhIFN-γ assisted by GroEL191～345 were as follows: refolding temperature 15℃, refolding time 4 h, pH 7.7, initial concentration of IFN-γ 100～200 mg/L and the molar ratio of GroEL 191～345 versus IFN-γ 1∶1～2∶1. Furthermore, the immobilization of GroEL191～345 on NHS-activated sepharose fast flow made it possible to be recycled. The protein yield and the specific activity of rhIFN-γ were 46.29% and 1.95×10⁷ U/mg respectively even the initial protein concentration was up to 400 mg/L.