During vertebrate development, neural crest cells migrate from the dorsal neural tube and give rise to pigment cells and most peripheral ganglia. To study these complex processes it is helpful to make use of in vitro techniques, but the transient and morphologically ill-defined nature of neural crest cells makes it difficult to isolate a pure population of undifferentiated cells. We have used several established techniques to obtain neural crest-containing cultures from quail embryos and have compared their subsequent differentiation. We confirm earlier reports of neural crest cell differentiation in vitro into pigment cells and catecholamine-containing neurons. However, our results strongly suggest that the 5-HT-containing cells that develop in outgrowths from thoracic neural tube explants are not neural crest cells. Instead, these cells arise from ventral neural tube precursors that normally give rise to a population of serotonergic neurons in the spinal cord and, in vitro, migrate from the neural tube. Therefore, results based on previously accepted operational definitions of neural crest cells may not be valid and should be reexamined. Furthermore, the demonstration that cells from the ventral (non-neural crest) part of the neural tube migrate in vitro suggests that the same phenomenon may occur in vivo. We propose that the embryonic "neural trough," as well as the neural crest, may contribute to the PNS of vertebrates.