Monoclonal antibodies to choline acetyltransferase (ChAT) were used in an immunocytochemical study to characterize putative cholinergic neurons and synaptic junctions in rat caudate-putamen. Light microscopy (LM) revealed that ChAT-positive neurons are distributed throughout the striatum. These cells have large oval or multipolar somata, and exhibit three to four primary dendrites that branch and extend long distances. Quantitative analysis of counterstained preparations indicated that ChAT-positive neurons constitute 1.7% of the total neuronal population. Electron microscopy (EM) of immunoreactive neurons initially studied by LM revealed somata characterized by deeply invaginated nuclei and by abundant amounts of organelle-rich cytoplasm. Surfaces of ChAT-positive neurons are frequently smooth, but occasional somatic protrusions and dendritic spines occur. Although infrequently observed, axons of ChAT-positive neurons branch, receive synapses, and become myelinated. Unlabeled boutons make both symmetrical and asymmetrical synapses with ChAT-positive somata and proximal dendrites, but are more numerous on distal dendrites. In addition, some unlabeled terminals form asymmetrical synapses with ChAT-positive somata and dendrites that are distinguished by prominent subsynaptic dense bodies. Light microscopy demonstrated a dense distribution of ChAT-positive fibers and punctate structures in the striatum, and these structures appear to correlate, respectively, with labeled preterminal axons and presynaptic boutons identified by EM. ChAT-positive boutons contain pleomorphic vesicles, and make symmetrical synapses primarily with unlabeled dendritic shafts. Furthermore, they establish synaptic contacts with somata, dendrites and axon initial segments of unlabeled neurons that ultrastructurally resemble medium spiny neurons. These observations, together with the results of other investigations, suggest that medium spiny GABAergic projection neurons receive a cholinergic innervation that is probably derived from ChAT-positive striatal cells. The results of this study also indicate that cholinergic neurons within caudate-putamen belong to a single population of cells that have large somata and extensive sparsely spined dendrites. Such neurons, in combination with dense concentrations of ChAT-positive fibers and terminals, are the likely basis for the large amounts of ChAT and acetylcholine detected biochemically within the neostriatum.