The expression, purification, detection, and assay of recombinant proteins have been made more convenient and rapid by the use of small affinity tags. To facilitate the purification of interferon-alpha2c (IFN-alpha2c) by metal chelate affinity chromatography, N-terminal 6-histidine tag was introduced via genetic manipulation. Two preparations of IFN material were purified; one contained IFN-alpha2c with the 6-histidine tag, and the other contained IFN-alpha2c without the 6-histidine tag. The antigenic properties of the human IFN-alpha2c subvariant with and without the 6-histidine tag, as well as the effects of the N-terminal 6-histidine tag on IFN-alpha2c interaction with the extracellular domain of human IFN-alpha receptor chain 2 (IFNAR2-EC) were examined. For the purposes of this study, IFNs were characterized by Western blots with anti-IFN monoclonal antibodies (mAb) and bioassays. Immunoblot analyses showed differences between IFN-alpha2c-6-histidine tag and IFN-alpha2a, b, c in their interaction with IFNAR2-EC. We also observed differences between IFN-alpha2c-6-histidine tag and IFN-alpha2a, b, c in bioactivities. This study is the first report that shows that an N-terminal 6-histidine tag on IFN-alpha2c can affect its interaction with receptor and cause a different bioactivity.