The human interferon-alpha 2 subvariants 2a, 2b and 2c differ by only one or two amino acids at positions 23 and/or 34 of the mature protein. In this study, the coding regions of the three interferon-alpha 2 subvariants were derived from the cDNA of interferon-alpha 2c by site-directed in vitro mutagenesis. The interferon-alpha subvariants were synthesized using the same Escherichia coli strain for production and were subsequently purified. Comparative studies revealed that they differ significantly in their biological and antigenic properties. Therefore, amino acid positions 23 and 34 seem to be crucial for structure/function of human interferon-alpha. Furthermore, the study points to the importance of defining, whether such minor structural variants of naturally occurring polypeptides represent functional variants.