In this study, the pharmacokinetic and pharmacodynamic properties of Lys³⁵, Met^N-terminal, and Lys¹⁷-mono-PEGylated recombinant human granulocyte colony stimulating factor (rhG-CSF) positional isomers were evaluated in rats. The in vitro biological activities of Lys³⁵, Met^N-terminal, and Lys¹⁷-mono-PEGylated rhG-CSF were determined by examining NFS-60 cell proliferation. Plasma concentrations of rhG-CSF and white blood cell (WBC) counts and absolute neutrophil conunt (ANC) were measured and pharmacokinetic and pharmacodynamic properties were determined after a single subcutaneous administration of the Lys³⁵, Met^N-terminal, or Lys¹⁷ isomers at 0.1 mg/kg in rats. The in vitro biological activities of Lys³⁵, Met^N-terminal, and Lys¹⁷-mono-PEGylated rhG-CSF individual positional isomers were 20.1%, 37.4%, and 15.3%, respectively, that of rhG-CSF. However, all three mono-PEGylated rhG-CSF isomers had a greater blood half-life ( T _1/2) and in vivo efficacy as determined by WBC counts and ANC than rhG-CSF, but no significant difference was observed between the three isomers. In conclusion, Lys³⁵, Met^N-terminal, and Lys¹⁷-mono-PEGylated rhG-CSF individual positional isomers exhibit an enhanced the in vivo pharmacokinetics and pharmacodynamics. Furthermore, three isomers have comparable in vivo pharmacokinetic and pharmacodynamic properties, but their in vitro biological activities are PEGylation site dependent.