Two human recombinant lymphoblastoid interferon-alpha subtypes, LyIFN-B (alpha 8) and LyIFN-D (alpha 1), and 10 hybrids generated therefrom were produced in Escherichia coli and purified. The antiviral and antiproliferative activities and the induction of (2'-5')oligoadenylate synthetase were compared to their receptor binding affinities. The IFN subtypes and their hybrids had similar specific antiviral activities on bovine cells. On human cells both the specific antiviral and antiproliferative activities of LyIFN-B were about 30-fold higher than those of LyIFN-D. This difference in activity could be attributed partly to the N-terminal amino acids 1 to 60 and partly to amino acids 61 to 92. A third domain affecting the biological activities was found within the carboxy-proximal segment from amino acids 93 to 150. The differences in these activities were found to correlate with their ability to bind the receptor, suggesting that the differences in activity might be due to altered binding of the IFNs to the cellular receptors. In contrast, the induction of (2'-5')oligoadenylate synthetase did not follow the same activity profile. On mouse cells, the efficiency of the hybrids was affected by at least four sites on the IFN protein. A hybrid with the N-terminal segment 1 to 60 from IFN-B and amino acids 61 to 166 from IFN-D had a specific antiviral activity on mouse cells as high as on human cells corresponding to a 500- and 5000-fold increase in specific activity compared to IFN-D and IFN-B, respectively. We suggest that on mouse cells the IFN activity may be more dependent on conformational differences than on human cells, which in turn might reflect a less precise fit to the mouse receptor than to the human receptor.