To investigate the structural basis for human interferon gamma (huIFNγ) binding to intracellular regions of the human IFNγ receptor (huIFNγR), we have subcloned and expressed the huIFNγR free of fusion proteins in the yeast strainPichia pastoris.HuIFNγ bound to the cytoplasmic domain of the receptor via the IFNγ C-terminus. Binding was inhibited by both human and mouse C-terminus peptides. N-terminus peptides failed to inhibit cytoplasmic binding. Thus, while extracellular receptor domain binding is species specific, binding to the cytoplasmic domain of the receptor is species non-specific. In solid-phase binding assays, IFNγ had a K_dof 3.7 × 10⁻⁸M for the newly expressed cytoplasmic domain. Peptide competitions showed that IFNγ bound to a receptor site corresponding to the membrane proximal residues 253-287, which is adjacent to the site of binding of the tyrosine kinase JAK2. The cytoplasmic binding affinity and binding site specificity suggest that the huIFNγR cytoplasmic domain can function independent of the extracellular domain to bind huIFNγ and induce the biological activity previously associated with internalized huIFNγ.