Metabotropic glutamate receptor 4 (mGluR4) is localized mainly to presynaptic membranes in the brain. Rat neostriatum has been reported to contain two types of mGluR4-immunoreactive axon varicosities: small, weakly immunoreactive varicosities that were distributed randomly (type 1) and large, intensely immunoreactive ones that were often aligned linearly (type 2). In the present study, most type 1 terminals formed asymmetric synapses on dendritic spines, whereas type 2 terminals made symmetric synapses on dendritic shafts, showing immunoreactivity for GABAergic markers. After depletion of neostriatal neurons, type 2 but not type 1 varicosities were largely decreased in the damaged region. When medium-sized spiny neurons (MSNs) were labeled with Sindbis virus expressing membrane-targeted green fluorescent protein, mGluR4 immunoreactivity was observed on some varicosities of their axon collaterals in immunofluorescence and immunoelectron microscopies. Furthermore, type 2 varicosities were often positive for substance P but mostly negative for striatal interneuron markers and preproenkephalin. Thus, striatonigral/striato-entopeduncular MSNs are likely to be the largest source of type 2 mGluR4-immunopositive axon terminals in the neostriatum. Next, in the double-immunofluorescence study, almost all choline acetyltransferase (ChAT)-immunopositive and 41% of NK1 receptor-positive dendrites were heavily associated with type 2 mGluR4-immunoreactive varicosities. Neuronal nitric oxide synthase (nNOS)-positive dendrites, in contrast, seemed associated with only a few type 2 varicosities. Conversely, almost all type 2 varicosities were closely apposed to NK1 receptor-positive dendrites that were known to be derived from cholinergic and nNOS-producing interneurons. These findings indicate that the mGluR4-positive terminals of MSN axon collaterals selectively form synapses with neostriatal cholinergic interneurons. J. Comp. Neurol. 500:908–922, 2007. © 2006 Wiley-Liss, Inc.