Neuronal precursor cells present in dorsal root ganglia (DRG) during early development have been previously shown to differentiate in vitro to neurons, as characterized by morphology, cell surface antigens, and electrophysiological properties (H. Rohrer, S. Henke-Fahle, T. El-Sharkawy, H. D. Lux, and H. Thoenen, 1985, Embo J. 4, 1709-1714). In the present study the conditions necessary for the initial differentiation and long-term survival of these cells were established, and the neurotransmitter phenotype of the newly differentiated neurons was analyzed. Neuronal precursor cells isolated from chick DRG at Embryonic Day 6 (E6) were found to require the presence of a polyornithine substrate coated with either laminin or fibronectin for initial neurite production and long-term survival. Neurons were unable to develop on polyornithine alone or on polyornithine coated with BSA. The survival and neurite outgrowth from neuronal precursor cells was not affected by the presence of nerve growth factor (NGF) during the first 9 hr in culture. NGF also had no effect on the proportion of cells expressing the neuron-specific Q211 antigen. However, after this initial differentiation period the neurons did require the presence of a survival factor. The neurons could be maintained for at least 6 days in culture both in the presence of NGF and in the presence of brain-derived neurotrophic factor (BDNF). At saturating concentrations of both survival factors no additive effects could be observed, indicating a complete overlap of NGF- and BDNF-responsiveness. Although the same proportion of cells survived with either NGF or BDNF during the first 3 days in culture, survival decreased in the presence of BDNF but not in the presence of NGF during the following 3 days in culture. The loss of BDNF responsiveness in vitro was also observed in vivo. After 6 days in culture about 70% of the neurons expressed substance P immunoreactivity, and approximately the same proportion was positive for myelin-associated glycoprotein immunoreactivity. The neurons did not express properties of adrenergic neurons such as tyrosine hydroxylase immunoreactivity or norepinephrine uptake. These findings indicate that the neuronal precursor cells from E6 DRG acquire the same characteristics in vitro as in their normal in vivo environment.