Two recombinant human granulocyte colony-stimulating factor (rhG-CSF) isoforms were isolated from the medium conditioned by an engineered Chinese hamster ovary (CHO) cell line. The two rhG-CSFs were characterized and were found to differ in the carbohydrate structure attached to Thr-133. The glycoform, referred to as Peak 1, contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)GalNAc; the Peak 2 glycoform contains the O-linked glycan Neu5Ac(alpha 2-3)Gal(beta 1-3)[Neu5Ac(alpha 2-6)]GalNAc. The two glycoforms displayed a similar biological activity in cultures of a mouse 32D C13 cell line and human bone-marrow myelo-monocytic progenitor cells (CFU-GM). In the latter test both glycoforms displayed a higher activity than nonglycosylated rMet-hG-CSF from Escherichia coli. The pharmacokinetic profile and activity of the two rhG-CSF glycoforms and of a mixture of them (Pool) were investigated in mice treated with a single injection of rhG-CSF at the doses of 125 micrograms and 250 micrograms/kg, given via the intravenous (i.v.) and the subcutaneous (s.c.) route, respectively. The plasma concentration profiles obtained were similar for all three substances and did not show any relevant differences in absorption or elimination. The pharmacokinetic parameters indicate that the three substances have similar area under the curve (AUCs), volumes of distribution, and terminal half-life. Furthermore, our data indicate a high bioavailability of the two different glycoforms of rhG-CSF when given to mice via the s.c. route either singularly or as a mixture. Detectable levels of rhG-CSF persisted for more than 8 h in the i.v. and more than 24 h in the s.c. route of administration. All three substances induced early neutrophilia in mice. All rhG-CSF-treated mice developed a two-four-fold rise in neutrophil counts as early as 4 h after the intravenous and 2 h after the subcutaneous injection. Relatively high levels of neutrophils were maintained for at least 8 and 24 h after i.v. and s.c. administration, respectively.