Granulocyte/macrophage colony-stimulating factor (GM-CSF) is an acidic glycoprotein that stimulates hematopoiesis in vitro and in vivo. Despite a high degree of sequence homology, the GM-CSFs from human and murine sources fail to crossreact in their respective colony-forming assays. On the basis of this rinding, a series of hybrid molecules containing various proportions of human-and murine-specific amino acid sequences were generated by recombinant DNA techniques and assayed for species-specific activity against human and murine marrow target cells. Two regions of GM-CSF, residues 38-48 and residues 95-111, were found to be critical for hematopoietic function. These regions are structurally characterized by an amphiphilic helix and by a disulfide-bonded loop, respectively, and are homologous in position in the human and murine growth factors. In addition, competition assays suggested that, together, these regions bind to the GM-CSF receptor.