BACKGROUND: Differential attachment, chemicals, and immunoaffinity absorption are frequently used to purify olfactory ensheathing cells (OECs). Although purity is high (> 90%), the complex process, high cost, decreased cell activity, and cell loss limit their application. OBJECTIVE: To purify OECs using differential attachment, cytosine arabinoside (Ara-C), and mitogen stimulation, and to analyze the biological characteristics of OECs. DESIGN, TIME AND SETTING: Molecular biology experiment of cell morphology and immunocytochemistry. The study was performed at the Institute of Neuroscience, Kunming Medical College between January 2005 and January 2007. MATERIALS: N2 was purchased from Gibico, USA; basic fibroblast growth factor (bFGF) from Invitrogen, USA; PCR master mix kit from Fermentas, USA; nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) from Santa Cruz Biotechnology, USA; OEC specific immunological marker NGF receptor (p75NGFR) from ABCAM, UK; immunological markers of oligodendrocyte and Schwann cells, cyclic nucleotide 3' phosphohydrolase (CNPase), from NeoMarkers, USA; inverted fluorescence microscope from Leica, Germany. METHODS: OECs were isolated from olfactory bulbs of mice provided by Institute of Cancer Research (ICR mice) at postnatal 1-2 days, and purified by differential attachment, Ara-C inhibition (5 mg/L), and 10 mu g/L mitogen bFGF and 0.5% N(2) stimulation. MAIN OUTCOME MEASURES: OEC growth was observed under inverted microscope; cell purity, as well as expression of NGF and BDNF, was determined by means of immunocytochemistry; expression of beta-NGF, BDNF, NT-3, platelet-derived growth factor-B (PDGF-B), bFGF, epidermal growth factor (EGF), NGF receptor TrkA, BDNF receptor TrkB, and NT-4 mRNA were detected by RT-PCR. RESULTS: The majority of in vitro cultured OECs was bipolar or tripolar, and purity was estimated to be > 92.4%. Immunocytochemistry demonstrated expression of p75NGFR, NGF, BDNF and CNPase. The RT-PCR results suggested that OECs expressed beta-NGF BDNF, NT-3, PDGF-8, bFGF, EGF, TrkA, and TrkB mRNA. CONCLUSION: Results demonstrated that purity of OECs was high, and that OECs expressed CNPase proteins and produced neurotrophic factors.