Previous studies using synthetic peptides and monoclonal antibodies have implicated the N-terminal 39-residue segment as a receptor binding region of mouse γ-interferon (MuIFNγ). In this work, we report the solution structure of this fragment (dissolved in water with 40% trifluoroethanol) as determined by proton NMR spectroscopy. The proton sequence-specific assignments were determined from TOCSY and NOESY spectra using established procedures. The secondary structure is characterized by two well-defined α-helical regions composed of residues 5–16 and 22–37. These two helices are joined by a loop. No NOESY contacts between the two helical regions were detected. Molecular models consistent with the NMR data were generated for MuIFNγ(1–39) using distance geometry and restrained molecular dynamics/energy minimization calculations. Comparison with similar N-terminal domains in the published NMR and crystallographic studies on the dimeric human and rabbit IFNγ suggests some similarities in the structures except that the helical regions in the fragment are longer, and considerable variation may exist in the relative orientation of the two helices in the solution phase. The presence of stronger αN sequential NOE's suggests that this peptide is flexible. The absence of NOESY contacts involving the N-terminal tripeptide suggests that this region undergoes rapid segmental motion. The data presented here on MuIFNγ(1–39), combined with the studies on human and rabbit IFNγ, suggest that the N-terminal receptor binding domain of the protein can undergo structural changes, the understanding of which may provide insight into the basis for receptor interaction by this lymphokine. © 1993, American Chemical Society. All rights reserved.