BACKGROUND: Midbrain-derived neural stem cells (mNSCs) can differentiate into functional mature dopaminergic neurons. The mNSCs are considered the ideal choice for cell therapy of Parkinson's disease. OBJECTIVE: To isolate rat embryonic mNSCs and to observe the differentiation characteristics of mNSCs induced by cell growth-promoting factors. DESIGN TIME AND SETTING: An in vitro cell culture study based on the molecular biology of nerve cells was carried out at the Institute of Clinical Medicine, China-Japan Friendship Hospital (China) from March to November 2007. MATERIALS: Sprague Dawley rats at embryonic day 14 were used in this study. Nestin antibody, beta -III glial fibrillary acidic protein (GFAP) antibody and cyclic nucleotide 3'-phosphohydrolase tubulin antibody, (CNPase) antibody were provided by Abeam; DMEM/F 12 medium and NZ supplement were provided by Invitrogen; epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF2) were provided by R&D Systems. METHODS: The ventral mesencephalon was dissected from embryonic day 14 rat embryos. By trypsin digestion and mechanical separation, the brain tissue was triturated into a fine single-cell suspension. The cells were cultured in 5 mL serum-free medium containing DMEM/F12, 1% N-2 supplement, 20 ng/mL EGF and FGF2. The mNSCs at the third generation were coated with 10 mu g/mL polylysine and induced to differentiate in the DMEM/F12 supplemented with 1% fetal bovine serum and 1% N-2. MAIN OUTCOME MEASURES: The neural spheres of the third passage were identified by nestin immunofluorescence; at the same time, the cells were induced to differentiate, and the types of differentiated cell were identified by immunofluorescence for beta III tubulin, GFAP and CNPase. RESULTS: Seven days after primary culture, a great many neurospheres could be obtained by successive pasage. Immunofluorescence assays showed that the neurospheres were nestin positive, and after differentiation, the cells expressed GFAP, CNPase and beta-III-tubulin. CONCLUSION: Embryonic day 14 rat mNSCs can differentiate into neuron-like cells and glial cells following induction by EGF, FGF2 and NZ additive.