Choline acetyltransferase (ChAT) immunocytochemistry and lectin-conjugated horseradish peroxidase (WGA-HRP) histochemistry were combined at the electron microscopic level to examine the morphology of cholinergic terminals in the canine centrum medianum-parafascicular complex (CM-Pf) and to localize cholinergic terminals making synaptic contact with retrogradely labeled CM-Pf thalamostriatal projection neurons. Following WGA-HRP injections into the caudate nucleus, CM-Pf neurons were heavily labeled with WGA-HRP reaction product. Examination with the electron microscope revealed retrogradely labeled neurons characterized by a large nucleus with deep infoldings of the nuclear envelope. ChAT-positive terminals were observed arising from small-diameter nonmyelinated axonal profiles. These terminals varied in size from 0.5 to 1.4 micron in long diameter. The smaller terminals (0.5-0.7 micron) were seen most frequently and established symmetrical or slightly asymmetrical synaptic contacts with small dendritic profiles. The larger ChAT-positive terminals (1.0-1.4 micron) were less frequently observed, contained several mitochondria and small clusters of pleomorphic vesicles, and contacted large dendritic shafts and cell somata. Some of the postsynaptic targets of both smaller and larger ChAT-positive terminals were identified as belonging to retrogradely HRP-labeled thalamostriatal neurons. These observations indicate that at least some thalamostriatal neurons within the CM-Pf complex are innervated by cholinergic terminals which probably arise from ChAT-positive cell bodies located within the pontomesencephalic tegmentum, particularly within the nucleus tegmenti pedunculopontinus and the laterodorsal tegmental nucleus. These findings provide evidence for direct influence by cholinergic brainstem nuclei over activities of thalamostriatal neurons.