Cholinergic neurons in the monkey neostriatum were examined at the light and electron microscopic level by immunohistochemical methods in order to localize choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. At the light microscopic level a sparse distribution of cholinergic neurons was identified throughout the caudate nucleus. Neurons had large (25-30 microns) somata, eccentric invaginated nuclei, primary dendrites of unequal diameters, and varicosities on distal dendritic branches. Ultrastructural study showed that the cholinergic cells had a cytoplasm abundant in organelles. Within dendritic branches, mitochondria and cisternae were localized primarily to varicosities. Synaptic inputs were distributed mostly to the dendrites and at least four types that formed symmetric or asymmetric synapses were observed. Immunoreactive fibers were relatively numerous within the neuropil and exhibited small diameters (0.1-0.15) micron) and swellings at frequent intervals. Cholinergic boutons that formed synapses were compared to unlabeled terminals making asymmetric synapses with dendritic spines. Results showed that ChAT-positive axons had significantly smaller cross-sectional areas, shorter synaptic junctions, and a higher density and surface area of mitochondria than the unlabeled boutons. Cholinergic axons formed symmetric synapses mostly with dendritic spines (53%) and the shafts of unlabeled primary and distal dendrites (37%). A relatively small proportion of the boutons contacted axon initial segments (1%) and cell bodies (9%) that included medium-sized neurons with unindented (spiny) and indented (aspiny) nuclei. The majority of dendritic spines contacted by cholinergic axons were also postsynaptic to unlabeled boutons forming asymmetric synapses. The results suggest that cholinergic neurons in the primary neostriatum belong to a single morphological class corresponding to the large aspiny (type II) interneuron identified in previous Golgi studies. Present results along with earlier Golgi-electron microscopic observations from this laboratory suggest that neostriatal cholinergic cells integrate many sources of intrinsic and extrinsic inputs. The observed convergence of ChAT-immunoreactive boutons and unlabeled axons onto the same dendritic spines suggests that intrinsic cholinergic axons modulate extrinsic inputs onto neostriatal spiny neurons at postsynaptic sites close to the site of afferent input.