The orthopoxviruses ectromelia virus (ECTV) and vaccinia virus (VACV) express secreted gamma interferon binding proteins (IFN-γBPs) with homology to the ligand binding domains of the host's IFN-γ receptor (IFN-γR1). Homology between these proteins is limited to the extracellular portions of the IFN-γR1 and the first ∼200 amino acids of the IFN-γBPs. The remaining 60 amino acids at the C termini of the IFN-γBPs contain a single cysteine residue shown to be important in covalent dimerization of the secreted proteins. The function of the remaining C-terminal domain (CTD) has remained elusive, yet this region is conserved within all orthopoxvirus IFN-γBPs. Using a series of C-terminal deletion constructs, we have determined that the CTD is essential for IFN-γ binding despite having no predicted homology to the IFN-γR1. Truncation of the ECTV IFN-γBP by more than two amino acid residues results in a complete loss of binding activity for both murine IFN-γ and human IFN-γ (hIFN-γ), as measured by surface plasmon resonance (SPR) and bioassay. Equivalent truncation of the VACV IFN-γBP resulted in comparable loss of hIFN-γ binding activity by SPR. Full-length IFN-γBPs were observed to form higher-ordered structures larger than the previously reported dimers. Mutants that were unable to bind IFN-γ with high affinity in SPR experiments failed to assemble into these higher-ordered structures and migrated as dimers. We conclude that the unique CTD of orthopoxvirus IFN-γBPs is important for the assembly of covalent homodimers as well as the assembly of higher-ordered structures essential for IFN-γ binding. Copyright © 2006, American Society for Microbiology. All Rights Reserved.