The development of central serotonergic neurons has been examined immunocytochemically utilizing an antiserum to serotonin (5-HT). Cells of the B4-B9 complex are first detected early on embryonic day 13 (E13; 7 mm crown rump length, CRL) and increase rapidly in number through E15 when they appear as bilateral columns situated from just caudal to the mesencephalic flexure to the pontine flexure. Aggregation of cells into subgroups is apparent soon after 5-HT neurons leave the ventricular zone, allowing the identification of certain subdivisions of the B4-B9 complex long before they assume their adult locations. The initial detection of 5-HT immunoreactive cells in the medulla occurs 1-2 days after the appearance of cells in the B4-B9 complex, although it has been reported that the time of origin of medullary raphe neurons (B1-B3) occurs before that of raphe neurons in the midbrain and pons (B4-B9). The first medullary 5-HT neurons, comprising the B3 subdivision occur ventro-laterally on E14 (10-11 mm CRL) at least 1-2 days before midline 5-HT neurons are visualized in the B1 and B2 groups. Thus, in contrast to cells in the B4-B9 complex, medullary 5-HT neurons complete much of their migration before they can be detected immunocytochemically, indicating that the time of onset of transmitter synthesis and storage may differ during differentiation of cells sharing a common neurotransmitter phenotype. The formation of ascending 5-HT fiber projections occurs rapidly from cells of the B4-B9 complex. Within 24 hours after the initial detection of 5-HT fiber immunoreactivity in such cells at E13, their axons are seen entering the caudal diencephalon (E14). These fibers have traversed the diencephalon and floor of the telencephalon by E15-E16 and reach the frontal neocortical pole by E17. The main ascending bundle of 5-HT axons courses through the diencephalon in the vicinity of the medial forebrain bundle, although some fibers also diverge and travel along certain pre-existing non-5HT pathways. However, examples are also found of acute directional changes in 5-HT fiber growth which do not appear to be associated with pre-formed non-5HT pathways. The pattern of ascending fiber outgrowth suggests a priority routing system which provides certain regions with 5-HT axons in a preferential sequence irrespective of the distance of these areas from 5-HT cell groups or from major bundles of ascending 5-HT fibers.