ABSTRACT Conjugates of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) attached to polyethylene glycol (PEG) chains were prepared using amine-reactive chemistry. Molecular masses of the PEGs were 20, 30, and 40 kDa. The monopegylated forms were isolated by anion-exchange chromatography and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), size-exclusion chromatography, mass spectrometry, reverse-phase high-performance liquid chromatography (HPLC), peptide mapping, in vitro cell proliferation bioassays, and rat pharmacokinetic studies. The pegylation site of the purified monopegylated products was identified as the N-terminus of the protein. All forms of pegylated GM-CSF were able to stimulate TF-1 cell proliferation in a colorimetric bioassay at concentrations equal to or lower than that of GM-CSF. Pharmacokinetic studies in rats demonstrated 32-fold, 27-fold, and 40-fold extensions in elimination half-lives for 20, 30, and 40 kDa PEG-GM-CSF, respectively, as compared with nonmodified GM-CSF.