We tested the hypothesis that 5-HT promotes the differentiation of enteric neurons by stimulating a developmentally regulated receptor expressed by crest-derived neuronal progenitors. 5-HT and the 5-HT₂ agonist (±)-2,5-dimethoxy-4-iodoamphetamine^.HCl (DOI) enhanced in vitro differentiation of enteric neurons, both in dissociated cultures of mixed cells and in cultures of crest-derived cells isolated from the gut by immunoselection with antibodies to p75^NTR. The promotion of in vitro neuronal differentiation by 5-HT and DOI was blocked by the 5-HT_1/2 antagonist methysergide, the pan-5-HT₂ antagonist ritanserin, and the 5-HT_2B/2C-selective antagonist SB206553. The 5-HT_2A-selective antagonist ketanserin did not completely block the developmental effects of 5-HT. 5-HT induced the nuclear translocation of mitogen-activated protein kinase. This effect was blocked by ritanserin. mRNA encoding 5-HT_2A and 5-HT_2B receptors was detected in the fetal bowel (stomach and small and large intestine), but that encoding the 5-HT_2C receptor was not. mRNA encoding the 5-HT_2B receptor and 5-HT_2B immunoreactivity were found to be abundant in primordial [embryonic day 15 (E15)–E16] but not in mature myenteric ganglia. 5-HT_2B-immunoreactive cells were found to be a subset of cells that expressed the neuronal marker PGP9.5. These data demonstrate for the first time that the 5-HT_2B receptor is expressed in the small intestine as well as the stomach and that it is expressed by enteric neurons as well as by muscle. It is possible that by stimulating 5-HT_2Breceptors, 5-HT affects the fate of the large subset of enteric neurons that arises after the development of endogenous sources of 5-HT.