Tile extract of E.coli containing recombinant human inteferon- (rhIFN-) with 7.0 mol/L guanidine hydrochloride ((Gu’IICl) was directly injected into a column of reverse phase liquid chromatography (RPLC) to separate and purify rhIFN- with acetic acid-water as mobile phase. Gu.HCI and most impure protcins call be separated by this way. Compared with the usual dilution method, the bioactivity recovery of tile purified rhIMN- was found to be over 500%. In addition. compared to common organic solvents employed in RPLC, acetic acid has higher freezing point. and therefore, it is easy to concentrate tile aim-protein by freeze-drying when acetic acid-water is used as Inobile phase in RPLC.