An extracellular domain of human granulocyte-colony stimulating factor (G-CSF) receptor was expressed in and purified from Chinese hamster ovary cells. Complex formation between G-CSF and the receptor was studied by size exclusion chromatography, followed by chemical cross-linking. The receptor-ligand complex contained an equimolar ratio of each protein. Crosslinking experiments using disucciniimide suberate revealed that the native complex contained at least two types of cross-linked complexes; one form contained one or two G-CSF molecules per receptor molecule, whereas another form contained one or two G-CSF per two receptor molecules. The tryptic peptide map of the cross-linked complex provided a unique peptide peak which was not found in a peptide map of the original protein. Sequence analysis and mass spectrometry of the peptide indicated that two peptides were covalently linked by cross-linker, one peptide from G-CSF and the other from the receptor. In the cross-linked peptide, Lys-242 of the receptor cross-linked the amino terminal Met of G-CSF through the cross-linker. It was also shown that the N-terminal Met of G-CSF was readily acetylated in the receptor-ligand complex, indicating that it was not directly involved in receptor binding. The results show that the N-terminal Met of G-CSF is located at a distance of approximately 11 A from a reactive Lys-242 of the receptor in the ligand-receptor complex.