AbstractMouse granulocyte-macrophage colony-stimulating factor (mGM-CSF) proteins with substitutions for residues located withinα-helix D were examined for biological activity and receptor binding properties. Alanine substitutions of the surface exposed positions indicated that several residues contribute to the ligand-receptor interface. Position K108and particularly D102appeared to dominate the binding epitope recognized by mGM-Rα. Several amino acid substitutions were made for K108which reduced binding with concommitant losses in bioactivity. Substitutions for D102resulted in binding affinities less than 0.1% that of the wild-type mGM-CSF and bioactivity decreased to 1.0%. Comparative analysis using high and low affinity binding conditions indicated that mGM-Rβbinding was unaffected by these mutations.