The caudal ventrolateral medulla (CVL) contains neurons that are vasodepressor and are a critical component of the baroreceptor reflex pathway. While electrophysiological studies suggest that CVL neurons are intercalated in the baroreceptor pathway between the nucleus tractus solitarius (NTS) and the rostral ventrolateral medulla (RVL), there is no direct evidence for this projection. Therefore, we identified CVL neurons that project to RVL by retrogradely labelling them with wheat germ agglutinin-apo-horseradish peroxidase conjugated to colloidal gold (WAHG) injected into the RVL. Retrogradely labelled neurons were seen in previously identified vasodepressor areas of the rostral CVL that are critical for the baroreceptor reflex. Double labelling for WAHG and tyrosine hydroxylase (TH) immunocytochemistry indicated that CVL neurons that project to the RVL (CVL --> RVL neurons) are distinct from the noradrenergic neurons of the A1 cell group. To establish the presence of a direct projection from the NTS to CVL --> RVL neurons, the retrograde tracer WAHG was pressure injected into the RVL and the anterograde tracer biocytin was iontophoresed into the NTS of anesthetized rats. After 4-6 h, anesthetized rats were perfused transcardially with 3.75% acrolein in 2% paraformaldehyde and sections through the CVL were processed for both markers. By light microscopy, numerous biocytin-labelled varicose processes overlapped neurons containing WAHG in the CVL. By electron microscopy, biocytin was found in myelinated and unmyelinated axons and in axon terminals (0.9 + 0.02 microns) that contained primarily small clear vesicles. These terminals formed predominantly asymmetric synapses on large (1.5-6.0 microns in diameter) dendrites within the CVL. Some of the post-synaptic perikarya and large dendrites contained WAHG associated with lysosomes and multivesicular bodies, indicating that they belong to neurons which project to the RVL. We conclude that CVL --> RVL neurons are (a) distinct from A1 noradrenergic cells; (b) receive direct synaptic contacts from NTS efferent terminals; (c) are potently and monosynaptically excited (asymmetric synapses) by NTS efferent terminals. These data support the hypothesis that CVL neurons are intercalated between the NTS and the RVL in the baroreceptor reflex pathway.