BACKGROUND: The differentiation of neural stem cells (NSCs) is an important for NSCs to be applied in clinical treatment. And whether the neurons differentiated from NSCs can be connected with other neurons or not comes into being a critical problem. OBJECTIVE: To observe the effect of nerve growth factor (NGF) on the growth and differentiation of in vitro cultured NSCs, and on the formation and growth of axons. DESIGN, TIME AND SETTING: The cytology in vitro experiment was performed at the Equipment Center of China Medical University from June 2007 to December 2008. MATERIALS: Three 2-3 day male Sprague Dawley rats were used in this study. NGF was purchased from Peprotech. METHODS: NSCs were isolated from neonatal rats by enzyme digestion and mechanical separation. At the fourth passage, cell clone masses received nestin immunocytochemistry. Remaining cells were dispersed by mechanical separation. Monoclone NSCs were incubated by limiting dilution assay, and made into 108 L-1 monoplast suspension in complete medium. NSCs were assigned into 2 groups. NSCs in the control group were incubated in 10% fetal bovine serum (FBS). NSCs in the induction group were incubated in the 10% FBS+50 ?g/L NGF for 5-7 days. The five isolated neurons with positive expression of neuron specific enolase (NSE) were observed. The number of axons was measured through concentric circles (37.5 ?m and 75 ?m diameter) circling neurons to detect the length of long axon. MAIN OUTCOME MEASURES: Cultured cells were identified by nestin, NSE and glial fibrillary acidic protein (GFAP) immunohistochemistry to test the number of neurons, number and length of axons. RESULTS: Neurospheres were Nestin-positive and could differentiate into the NSE-positive or GFAP-positive cells. At day 6, the numbers of neurons and axons were significantly more, and the length of longest axons was significantly longer in the induction group than in the control group (t=3.301, 2.982, 4.012, P