The early development of serotonin-containing neurons was studied in wholemounts of the fetal rat brain (E12-E18). The wholemounts were treated immunocytochemically according to an immunoperoxidase technique to reveal a panorama of developing serotoninergic neurons. Serotoninergic neurons were localized to two discrete groups or clusters within the brainstem. Serotonin-containing neurons were identified first at E12 forming a rostral cluster of cells just caudal to the mesencephalic flexure. The more caudal cluster of cells first appeared at E14 in the medulla. During the period from E12 to E18, the immunoreactive cells increased in number and acquired a more complex dendritic tree while migrating to their permanent position. At E16, cells of the rostral group exhibited remarkably uniform mediolateral orientation. The rostral group of immunoreactive neurons gave rise to almost all ascending fibers, whereas the caudal group gave rise to the majority of descending fibers. Growing serotoninergic fibers were tipped by prominent growth cones, which were strongly immunoreactive. The fibers demonstrated prominent orientational selectivity with an almost total separation into ascending and descending bundles. Some of the ascending immunoreactive fibers displayed acute changes in their direction of growth, suggesting that the orientation of serotininergic fibers is mediated by directional cues that are specific to particular populations of serotoninergic fibers. Serotoninergic axons within the medial forebrain bundle were demonstrated particularly well and their ascent and rate of growth toward the forebrain could be easily followed. Immunoreactive fibers entered the telencephalon at E17 two portals, one along the lateral border of the hypothalamus and one rostrally, adjacent to the olfactory tubercle. In wholemounts at E18, fibers arising from this latter location could be followed as two distinct bands within the pallium; a basal band located ventrolaterally, adjacent to the lateral olfactory tract, and a dorsal band located at the medial edge of the telencephalon. Both fascicles were directed toward the occipital pole and contained unbranched fibers. At E18, serotoninergic axons arising from these two loosely organized fascicles covered most of the frontal telencephalon. The results of the present study indicate that wholemounts of embryonic brain can provide novel spaciotemporal data on the development of neuro-transmitter systems and may in the future prove to be useful experimental preparations in developmental neurobiology.