The effects of the urea concentration and methods of addition on refolding efficiencies were studied by use of lysozyme and carbonic anhydrase to attain high refolding efficiencies at high protein concentrations. Depending on the protein concentration to be refolded, a suitable urea concentration exists. Thus, by selecting such a urea concentration for formation of a so-failed loosely folded state of protein molecules without aggregate formation in renaturation mixtures, high refolding efficiencies above 80% were obtained in both lysozyme and carbonic anhydrase, A method of addition of denatured protein solutions to refolding buffers in fed-batch manner was devised to adjust the urea concentration in renaturation mixtures to follow the best trajectory for high refolding efficiencies through the course of refolding. By fed-batch addition of a denatured lysozyme solution to the refolding buffer containing 2.5 mol/L urea, a refolding efficiency of 85% was attained even at a lysozyme concentration of 7.3 kg/m(3), Urea in renaturation mixtures could be removed by dialysis without loss of the enzyme activities. These results are useful to attain a high efficiency at a high protein concentration in refolding processes.