Antibodies directed against the C-terminus of the rat vesicular acetylcholine transporter mark expression of this specifically cholinergic protein in perinuclear regions of the soma and on secretory vesicles concentrated within cholinergic nerve terminals. In the central nervous system, the vesicular acetylcholine transporter terminal fields of the major putative cholinergic pathways in cortex, hippocampus, thalamus, amygdala, olfactory cortex and interpeduncular nucleus were examined and characterized. The existence of an intrinsic cholinergic innervation of cerebral cortex was confirmed by both in situ hybridization histochemistry and immunohistochemistry for the rat vesicular acetylcholine transporter and choline acetyltransferase. Cholinergic interneurons of the olfactory tubercle and Islands of Calleja, and the major intrinsic cholinergic innervation of striatum were fully characterized at the light microscopic level with vesicular acetylcholine transporter immunohistochemistry. Cholinergic staining was much more extensive for the vesicular acetylcholine transporter than for choline acetyltransferase in all these regions, due to visualization of cholinergic nerve terminals not easily seen with immunohistochemistry for choline acetyltransferase in paraffin-embedded sections. Cholinergic innervation of the median eminence of the hypothalamus, previously observed with vesicular acetylcholine transporter immunohistochemistry, was confirmed by the presence of vesicular acetylcholine transporter immunoreactivity in extracts of median eminence by western blotting. Cholinergic projections to cerebellum, pineal gland, and to the substantia nigra were documented by vesicular acetylcholine transporter-positive punctate staining in these structures. Additional novel localizations of putative cholinergic terminals to the subependymal zone surrounding the lateral ventricles, and putative cholinergic cell bodies in the sensory mesencephalic trigeminal nucleus, a primary sensory afferent ganglion located in the brainstem, are documented here. The cholinergic phenotype of neurons of the sensory mesencephalic trigeminal nucleus was confirmed by choline acetyltransferase immunohistochemistry. A feature of cholinergic neurons of the central nervous system revealed clearly with vesicular acetylcholine transporter immunohistochemistry in paraffin-embedded sections is the termination of cholinergic neurons on cholinergic cell bodies. These are most prominent on motor neurons of the spinal cord, less prominent but present in some brainstem motor nuclei, and apparently absent from projection neurons of the telencephalon and brainstem, as well as from the preganglionic vesicular acetylcholine transporter-positive sympathetic and parasympathetic neurons visualized in the intermediolateral and intermediomedial columns of the spinal cord. In addition to the large puncta decorating motor neuronal perikarya and dendrites in the ventral horn, vesicular acetylcholine transporter-positive terminal fields are distributed in lamina X surrounding the central canal, where additional small vesicular acetylcholine transporter-positive cell bodies are located, and in the superficial layers of the dorsal horn. Components of the central cholinergic nervous system whose existence has been controversial have been confirmed, and the existence of new components documented, with immunohistochemistry for the vesicular acetylcholine transporter. Quantitative visualization of terminal fields of known cholinergic systems by staining for vesicular acetylcholine transporter will expand the possibilities for documenting changes in synaptic patency accompanying physiological and pathophysiological changes in these systems.