Immunocytochemistry was used to establish the cellular localization of choline acetyltransferase [ChAT] throughout the rostrocaudal portions of the nuclei of the solitary tracts [NTS] in rat brain. By light microscopy, two distinct populations of ChAT-positive cells were identified. The first consisted of relatively few, medium-sized neurons located in the caudal one-half of the medial NTS just dorsal to the dorsal motor nucleus of the vagus. The second population of ChAT-labeled neurons was located more anteriorly and surrounded the medial and dorsal borders of the tractus solitarius. These cells were more abundant and smaller diameter than those located more caudally. Thick, non-varicose processes with the light microscopic characteristics of dendrites also were selectively labeled for ChAT. A few of these processes were located near or were continuous with the labeled perikarya of the NTS. However, the vast majority of the immunoreactive processes could be traced from ChAT-labeled perikarya in the ventrally adjacent dorsal motor nucleus of the vagus. These dorsally directed dendrites aborized extensively throughout the NTS, but they were densest in the rostral two-thirds of the nucleus. Caudally, the labeled dendrites coursed horizontally, forming a commissure-like structure between the two vagal motor nuclei. Electron microscopy confirmed the perikaryal and dendritic localization of ChAT in the NTS. The perikarya were characterized by dense peroxidase immunoreactivity throughout the cytoplasm, infolded nuclear membranes, and somatic synapses. The labeled dendritic profiles also were intensely immunoreactive and received synaptic input from unlabeled terminals. The unlabeled afferents to somata and dendrites contained large populations of small clear vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)