One-step simultaneous purification and renaturation of interferon-alpha from the inclusion body of E. coli expreessed by high-performance hydrophobic interaction chromatography(HPHIC) are presented. The inclusion body was dissolved by 8 mol/L guanidine hydrochloride. The sample lysis was centrifuged and the solution was renaturated by three different methods, i.e. hydrophobic interaction chromatography, dilution and dialysis. The total activity yield renaturation by hydrophobic interaction chromatography was 2 times and 2.6 times as much as that by dilution and by dialysis. The purity of the target product was 95% according to high-performance size exclusion chromatography and the specific activity was 1.8 x 10(8) IU/mg. The method developed is simpler and more efficient than others.