Abstract Proteolytic digests of biologically active fractions of recombinant DNA-derived human granulocyte macrophage colony stimulating factor (rhGM-CSF) expressed in large quantities in Escherichia coli have been analyzed by fast atom bombardment mass spectrometry (FAB-MS) and high-performance liquid chromatography (HPLC). The major fraction fractionated by reversed-phase HPLC (RP-HPLC) was N-terminal methionine additional type of human GM-CSF. Disulfide bond linkages were confirmed between Cys-54 and Cys-96, Cys-88 and Cys-121. Furthermore five minor fractions were isolated, and characterized by FAB-MS and HPLC. One of them was a rhGM-CSF in which two amino acids at the N-terminal (Met-Ala) were deleted. Other minor fractions were rhGM-CSFs of which one of four methionine residues was sulfoxidated, respectively. These findings suggest that the removal of amino acids occurs post-translationally, and that the sulfoxidation of methionine residues occurs during oxidative renaturation and/or purification. FAB-MS combined with HPLC provides a very useful method for the characterization of recombinant proteins, because the method is very convenient and simple.