IFN-gamma is an essential immunoregulatory lymphokine for a variety of immunologic functions including upregulation of MHC Ag. The elucidation of the structure, particularly the receptor binding domains, should further enhance our understanding of its mechanism of action, and provide a rational basis for modulation of its activity by alteration of its structure. A predicted model of murine IFN-gamma structure has been constructed based on data derived from our synthetic peptide studies, circular dichroism spectra, and predictive algorithms for secondary structure, surface accessibility, and tertiary structure, as well as predicted structural similarities to IL-2. Direct synthetic peptide competition studies using five overlapping peptides that encompassed the entire IFN-gamma sequence of 133 amino acids showed that only the N-terminus of IFN-gamma, IFN-gamma(1-39), blocked binding to receptor, suggesting that the N-terminus is directly involved in receptor binding. Rabbit antibodies to the N-terminal (IFN-gamma(1-39)) and C-terminal (IFN-gamma(95-133)) peptides neutralized IFN-gamma activity, whereas antibodies to the three peptides that spanned the internal region, sequence 36-107, were without effect. Thus, the antibody data support the involvement of the N-terminus as a receptor binding domain and also suggest that the C-terminus of the molecule is also a binding domain. Predictive algorithms assign six alpha-helices and five turns to the molecule and circular dichroism spectra of both intact human and murine IFN-gamma and synthetic peptides of murine IFN-gamma showed that the molecule is mainly alpha-helical in structure. Drawing mainly on the four alpha-helix bundle model, a common motif in globular proteins such as IFN-gamma and IL-2 (whose crystalline structure is known), we constructed a simple model of the tertiary structure of IFN-gamma that fits well with our synthetic peptide and circular dichroism data. The model consists of the four-alpha-helical bundle along with N- and C-terminal helices that are predicted to be closely associated and together form the receptor binding domains. The model presented should contribute to further understanding the molecular basis of IFN-gamma action and allow us to begin modulating the function of IFN-gamma through the design of agonists and antagonists.