We developed an efficient inclusion body pro-urokinase refolding method from recombinant Escherichia coli. The protein was efficiently refolded when a heat treatment was applied to a protein denaturing solution containing guanidine hydrochloride. The total enzyme activity and the specific activity in response to the 50 degrees C heat treatment compared to normal method (25 degrees C) were enhanced about 10 and 25%, respectively. Moreover, enhanced protein refolding was also observed in the case of a reduced protein concentration in the protein refolding solution. The result indicates that correct protein folding is closely related to the protein concentration in the refolding solution.