Serotonergic neurons have previously been identified in the enteric nervous systems of humans, subhuman primates, rodents, and rabbits. The distribution of enteric serotonergic neurons in lower vertebrates was examined in order to determine if these neurons are restricted to mammals and, if they are found more generally amongst vertebrates, when they first appear in vertebrate phylogeny. Since mammalian enteric serotonergic neurons take up 3H-serotonin by a highly specific mechanism, the radioautographic demonstration of axonal uptake of 3H-serotonin was used as the primary tool in looking for these neurons. As controls, conditions known to interfere with 3H-serotonin uptake by mammalian enteric neurons were also examined. These controls included incubation with 10 microM fluoxetine, a specific antagonist, incubation in Na+-free medium, and incubation in the presence of a 100-fold excess of nonradioactive serotonin. Radio-autographic labeling had to be absent or greatly reduced under all three control conditions for labeling by 3H-serotonin to be considered specific. Labeled enteric axons were found in cyclostomes (hagfish), teleosts (goldfish), and amphibia (bull-frog) but not in tunicates (sea squirt; sea vase) or echinoderms (sea cucumber). In addition, the serotonin concentration was measured in the intestine of two vertebrates, hagfish and goldfish, that do not have serotonin-containing enterochromaffin cells. Serotonin was found in both; in hagfish, the amine concentration was highest in preparations of muscularis externa containing the myenteric plexus. It is concluded that enteric serotonergic neurons arose early in vertebrate evolution, possibly in an ancestral chordate resembling amphioxus, although probably not in more primitive prevertebrates, and that they are a general feature of the vertebrate bowel.