The crystal structure of human interferon (Hu-IFN)-alpha2b has been determined at 2.9 A resolution. This experimentally derived model provides an accurate structural scaffold on which amino acid changes between the different human IFN-alpha subtypes may be compared. Accurate structural data are essential to identify structurally important residues buried in the hydrophobic core of the molecule from solvent accessible residues that may participate in receptor binding. Furthermore, the location and chemical composition of each amino acid substitution may be used to predict potential conformation changes that may occur in the different subtypes. The possible structural and surface effects of these amino acid changes on receptor binding and biologic activity are analyzed in the context of a proposed IFN-alpha receptor complex model. This model can be improved and corrected as additional biochemical and experimental structural data are obtained. These modeling techniques have been used to assess the structural and functional consequences of amino acid changes between Hu-IFN-alpha2b and consensus IFN (IFN-con1), Hu-IFN-alpha8, and Hu-IFN-alpha1, which each have distinct receptor-binding and biologic properties. Amino acids in IFN-alpha1 and IFN-alpha8 were identified that may explain the lower specific activities of these subtypes versus the activity of IFN-alpha2b. In contrast, a molecular explanation for the reported differences between IFN-alpha2b in receptor binding affinity of either IFN-alpha8 or IFN-con1 was not readily apparent. Notably, 15 of the 19 amino acid differences in IFN-con1 compared with IFN-alpha2b are located on the exterior surface, where they may enhance the antigenicity of this synthetic, nonnaturally occurring IFN. These modeling studies should assist in the design of further experiments to clarify these observations.