Gamma interferon (IFN-gamma) production is important in the host response to, and recovery from, infection with Ectromelia virus (ECTV) and Vaccinia virus (VACV). The orthopoxviruses have evolved several mechanisms to subvert the IFN-gamma response. IFN-gamma-binding protein (IFN-gammaBP) is a virally encoded homologue of the host IFN-gamma receptor that blocks the effects of IFN-gamma in the infected host. Unlike the cellular receptors, whose ligand specificity is restricted to their own species, the orthopoxvirus IFN-gammaBPs bind IFN-gamma from several species. The reason for this relaxed specificity has yet to be explained. ECTV, a mouse pathogen, encodes an IFN-gammaBP that has been shown to inhibit the activity of both human and murine IFN-gamma (hIFN-gamma and mIFN-gamma, respectively). In contrast, the IFN-gammaBP from VACV is unable to inhibit mIFN-gamma, but retains activity against hIFN-gamma. To determine which region(s) in the ECTV sequence is responsible for its ability to bind to mIFN-gamma with high affinity, a series of chimeric IFN-gammaBPs, as well as individual point mutants in the ECTV sequence corresponding to the amino acid changes from the VACV sequence, were constructed. The affinities of the chimeric and point mutant IFN-gammaBPs for mIFN-gamma were tested by using surface plasmon resonance and bioassay. By using this strategy, several key residues in the ligand-binding domains of the ECTV sequence have been identified that are responsible for high-affinity binding to mIFN-gamma. Substitution of the ECTV residue at these positions in VACV resulted in a dramatic increase in the affinity of the VACV IFN-gammaBP for mIFN-gamma.