The varieties of alpha-amylase denatured with 8.0 mol/L urea and 7.0 mol/L guanidine hydrochloride (GuHCl) solutions and their refolded intermediates were separated and renatured through high performance hydrophobic interaction chromatography (HPHIC). With investigating and comparing the numbers of the refolded intermediates and the bioactivity recovery of renaturation by means of HPHIC, it was found that these are quite different. The results can be attributed to the different denaturation mechanism of alpha-amylase with the two denaturing agents. The numbers of the refolded intermediates of urea-unfolded alpha-amylase were found to be more than that denatured by GuHCl, because GuHCl may make the changes in the surface of alpha-amylase molecules, by contrast, urea may do the changes not only in the surface of the protein, but also in the hydrophobic packet interior of the molecules. The HPHIC packings with weak hydrophobicity was found to have good separation efficiency and to have almost continuous and many peaks denoting these intermediates, while that with strong hydrophobicity was found to have higher bioactivity recovery. The numbers of the intermediates of urea-unfolded alpha-amylase were much more than that of bovine pancreatic trypsin inhibitor (BPTI) separated by Weissman et al. The effect of temperature on the separations and the renaturations were investigated too. The result showed that the higher the column temperature, the more the refolded intermediates of unfolded alpha-amylase separated and the lower bioactivity recovery were obtained from HPHIC.