After being synthesized in the cytoplasm of axon terminals, acetylcholine is packaged into synaptic vesicles by a proton-dependent transporter, vesicular acetylcholine transporter (VAChT). Localization of VAChT is restricted to cholinergic neurons, especially their terminals. We used an anti-VAChT antibody from INCSTAR to localize cholinergic terminals in the rat cochlear nucleus (CN), an important brainstem auditory center. VAChT immunoreactivity in the rat CN appears as labeled puncta and a few connecting fibers. In ventral CN (VCN), VAChT-labeled puncta are closely associated with somatic profiles of medium to large neurons. In and near the granular regions of VCN, VAChT-labeled puncta are more diffusely scattered. In the subpeduncular corner and the medial sheet, some VAChT-labeled fibers are seen in connection with especially prominent VAChT-labeled puncta. In dorsal CN (DCN), VAChT-labeled puncta show no clear association with somata and are found in all layers. Ultrastructurally, VAChT labeling is seen in the cytoplasm and is associated with synaptic vesicle membrane of terminals with small round vesicles. Such VAChT-labeled terminals synapse with cell bodies and dendrites in the CN.(J Histochem Cytochem 47:83-90, 1998)