The precursor cells that form the enteric nervous system (ENS) are multipotent when they arrive in the gut from the neural crest. Their differentiation thus depends on signals from the enteric microenvironment. Crest-derived cells were isolated from the fetal rat bowel by immunoselection at E14 with NC-1/HNK-1 antibodies and secondary antibodies coupled to magnetic beads. NC-1/HNK-1- immunoreactive cells were enriched approximately 36-fold. The NC-1/HNK- 1-selected population and the residual population were plated at equal cell density and maintained in a defined medium for 6–7 d. The total number of cells found in the cultures of the residual cells was three- to fourfold that in cultures of immunoselected cells. Neurotrophin-3 (NT-3), but not nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), or neurotrophin-4/5 (NT-4/5), was found to increase the proportion of neurons (neurofilament-immunoreactive or neuron-specific enolase-immunoreactive) or glia (S-100-immunoreactive) (from 6.6 +/- 0.9% to 15.2 +/- 1.4%; p < 0.001). This effect was concentration dependent (from 1 to 40 ng/ml) and observed only in the cultures of immunoselected cells. NT-3 also enhanced neurite outgrowth. NT-3 increased neither cell number nor bromodeoxyuridine incorporation and thus was not mitogenic. Exposure of immunoselected cells to NT-3 rapidly and transiently induced the appearance of nuclear Fos immunoreactivity. Transcripts coding for TrkC, the transducing receptor for NT-3, were identified in the fetal rat gut (E14-E16) and in the immunoselected population of cells using reverse transcriptase and the polymerase chain reaction. It is concluded that NT-3 specifically promotes the differentiation of enteric crest-derived cells as neurons or glia and may thus play a role in the development and/or maintenance of the ENS.