The ventral striatum, previously defined as including the nucleus accumbens, substriatal grey, olfactory tubercle and striatal cell bridges has been examined in an immunocytochemical study with monoclonal antibodies to choline acetyltransferase (ChAT) in order to identify putative cholinergic neurons and synaptic junctions within the region. Light microscopy revealed ChAT-positive neurons with similar morphological characteristics in all divisions of ventral striatum. The somata of immunoreactive neurons were round or elongated in shape, approximately 10 X 21 microns in size and had two to four dendrites that coursed long distances and occasionally branched. Electron microscopy of ChAT-positive neurons in substriatal grey initially studied by light microscopy revealed that unlabelled boutons occasionally formed synapses with immunoreactive somata and proximal dendrites, but were more numerous along distal dendrites. Light microscopy demonstrated that ventral striatal neuropil contained numerous ChAT-positive fibres and punctate structures that varied in concentration from moderate to very dense. The lateral border of the substriatal grey and the area within, and adjacent to, all islands of Calleja exhibited the most dense ChAT-positive punctate staining. Additionally, the medial portion of nucleus accumbens was more densely ChAT-positive than the lateral, and the olfactory tubercle displayed laminar variations of immunoreaction product. Counterstained immunocytochemical specimens demonstrated that some areas of dense ChAT-positive punctate staining were associated with clusters of ChAT-negative, medium-sized neurons. Furthermore, electron microscopic observations of substriatal grey revealed that ChAT-positive dense regions were associated with numerous immunoreactive boutons, some of which established synapses with unlabelled somata, dendritic shafts and spines. These results suggest that the densely ChAT-positive neuropil areas within ventral striatum receive more cholinergic innervation than the more lightly stained neuropil areas. There are numerous similarities in the morphological characteristics of ChAT-positive neurons and synapses observed in ventral striatum when compared with those previously described in dorsal striatum. However, some differences were observed, such as smaller somal sizes in ventral, as contrasted with dorsal striatum, and a substantial variation in ChAT-positive fibre and punctate neuropil staining seen within the ventral but not the dorsal striatum. Such differences suggest that the ventral striatum may exhibit greater heterogeneity of cholinergic function than the dorsal striatum.