We compared the antigenic properties of human interferon-alpha2c (IFN-alpha2c), IFN-alpha21a, hybrids IFN-alpha21a/alpha2c, and their mutants, using a panel of 27 anti-IFN-alpha1, anti-IFN-alpha2, and anti-IFN-alpha8/1/8 monoclonal antibodies (mAb). After immunoanalysis by ELISA, we found parental IFN-alpha2c and IFN-alpha21a to be antigenically distinct. Lack of reactivity of anti-IFN-alpha1 mAb with IFN-alpha21a indicated an antigenic distinction between subtypes alpha1 and alpha21a. The antigenic properties of hybrid IFNs consisting of the N-terminal portion (1-75) of IFN-alpha21a and the C-terminal portion (76-166) of IFN-alpha2c were analyzed with mAb recognizing defined regions of IFN-alpha2c, IFN-alpha1, and IFN-alpha8/1/8. We found that extending the sequence of IFN-alpha21a up to position 95 in hybrid molecule decreased the immunoreactivity of mAb specific for the antigenic structure formed by residues --112-132-- (helix D) of IFN-alpha2c. Inserting the sequence 76-81 (loop BC) of IFN-alpha2c into the sequence of 1-95 of IFN-alpha21a restored the reactivity of anti-IFN-alpha2c mAb. Some amino acid substitutions at positions 86 and 90 (helix C) of hybrid IFN-alpha21a/alpha2c also affected the immunoreactivity of C-terminal-specific mAb, which recognize helix D, but did not influence the structure of C-terminus of IFN (aa 151-165). Changes in the structure of constructs affected not only their antiproliferative activity but also their antiviral activity on human cells.