The high-affinity choline transporter (CHT) is a protein integral to the function of cholinergic neurons in the central nervous system (CNS). We examined the ultrastructural distribution of CHT in axonal arborizations of the mesopontine tegmental cholinergic neurons, a cell group in which CHT expression has yet to be characterized at the electron microscopic level. By using silver-enhanced immunogold detection, we compared the morphological characteristics of CHT-immunoreactive axon varicosities specifically within the anteroventral thalamus (AVN) and the ventral tegmental area (VTA). We found that CHT-immunoreactive axon varicosities in the AVN displayed a smaller cross-sectional area and a lower frequency of synapse formation and dense-cored vesicle content than CHT-labeled profiles in the VTA. We further examined the subcellular distribution of CHT and observed that immunoreactivity for this protein was predominantly localized to synaptic vesicles and minimally to the plasma membrane of axons in both regions. This pattern is consistent with the subcellular distribution of CHT displayed in other cholinergic systems. Axons in the AVN showed significantly higher levels of CHT immunoreactivity than those in the VTA and correspondingly displayed a higher level of membrane CHT labeling. These novel findings have important implications for elucidating regional differences in cholinergic signaling within the thalamic and brainstem targets of the mesopontine cholinergic system.