The postsynaptic targets of cholinergic boutons in the rat neostriatum were assessed by examination in the electron microscope of boutons that were immunoreactive for choline acetyltransferase, the synthetic enzyme for acetylcholine. These boutons formed symmetrical synaptic specializations with neostriatal neurons. Of 209 immunoreactive synaptic boutons observed in random searches of the neostriatum, 45% made contact with dendritic shafts, 34% with dendritic spines, and 20% with neuronal perikarya. Many of the postsynaptic structures had ultrastructural characteristics of the most common type of striatal neuron, the medium-size densely spiny neuron. This was confirmed by the examination in the electron microscope of Golgi-impregnated medium-size spiny neurons from sections that had also been immunostained for choline acetyltransferase. Immunoreactive boutons formed symmetrical synaptic specializations with all parts of the neurons examined, i.e., perikarya, proximal and distal dendritic shafts, and dendritic spines. Two of the Golgi-impregnated medium-size spiny neurons that received input from the cholinergic boutons were also retrogradely labelled with horseradish peroxidase that had been injected into the substantia nigra, they were thus further characterized as striatonigral neurons. Similarly, seven retrogradely labelled perikarya of striatonigral neurons were found to receive input from the cholinergic boutons. It is concluded that cholinergic boutons in the neostriatum form synaptic specializations and that one of their major targets is the medium-size densely spiny neuron that projects to the substantia nigra. The topography of the cholinergic afferents of these cells is distinctly different from that of other boutons derived from local neurons and from boutons that form asymmetrical synaptic specializations, but it is similar to that of the dopaminergic boutons originating from neurons in the substantia nigra.