Site-directed in vitro mutagenesis was used to create analogs of human interferons (IFNs)-alpha 1 and -alpha 4. Analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate cell-free protein synthesis system. Amino acid substitutions for the highly conserved residues at positions 33, 121, 122 and 123 greatly reduced the antiviral and antiproliferative activities on human cells of IFNs-alpha 1 and -alpha 4. In general, the amino acid substitutions had much less effect on the antiviral activities on bovine, compared with human, cells. Substitutions at positions 31, 41, 42, 124, 134, 135 and 136 had little or no effect on the biological activities of the IFN analogs. The abrogation of antiviral activity resulting from amino acid substitutions for the arginine residue at position 33 suggests that this arginine residue is required for binding to the IFN-alpha receptor on the cell surface.