Interferon (IFN)-tau is a type I IFN that is responsible for the maternal recognition of pregnancy in ruminants. This protein also has classic IFN-like properties, including antiviral, antiproliferative, and immunomodulatory functions. Using IFN-tau as a model, we examined the structural basis for the activity of type I IFNs, focusing on amino acids within helix A and the first section of the AB loop, which have been proposed as a site for receptor interaction. Six amino-acid substitutions were made that replaced a residue in ovine IFN-tau1mod with the corresponding residue in human IFN-alphaA. Receptor binding was enhanced by a P26L mutation and was reduced by a conservative lysine-to-histidine substitution at residue 34. Alterations in the antiviral and antiproliferative activities of the IFN-tau mutants were not always correlated, but both functions were maintained or enhanced relative to the wild-type IFN-tau by the proline-to-leucine mutation at residue 26. In contrast, this mutation did not affect the low in vitro cytotoxicity that is characteristic of ovine IFN-tau1mod. Thus, the IFN-tau P26L mutant may have potential as an improved IFN-based therapeutic.