The effect of guanidinium chloride on protein unfolding was regarded as a combination of the effects on interactions such hydrophobic interaction or hydrogen bond, and ionic interaction that construct a higher-order structure of the protein. Urea and LiCl instead of guanidinium chloride were used for refolding of denatured reduced lysozyme by assuming that urea and LiCl individually control hydrophobic interaction or hydrogen bond and ionic interaction, respectively. The refolding media were prepared using various combinations of urea and LiCl concentrations. Reoxidation of thiol groups was carried out in a glutathione redox system. The recovered enzyme activity at a lysozyme concentration of 10 mu M was usually less than 20% in 50 mM Tris buffer due to irreversible misfolding and/or aggregation during refolding. In the presence of urea and LiCl at high concentrations the recovered activity increased markedly. Complete renaturation could be achieved in a broad range of concentrations of urea and LiCl around the optimum of 3 M urea and 1.5 M LiCl, while it could not be achieved solely with guanidinium chloride. The availability of the concept that multifunction of guanidinium chloride can be separated approximately into two monofunctions of urea and LiCl was confirmed by the optimum design of refolding media prepared by combinations of urea and LiCl. A characteristic diagram for the refolding of lysozyme was depicted. This diagram will aid the optimum design of refolding media and study of the refolding mechanism.