The subthalamic nucleus (STN) of the basal ganglia plays a crucial role in control of voluntary movements. STN neurons are well known to project excitatory efferents mainly to the external segment of the globus pallidus (GPe), entopeduncular nucleus and substantia nigra. Recently, we unraveled that the vast majority of rat STN neurons sent axon collaterals not only to these target nuclei but also to the neostriatum, suggesting that the neostriatum is a novel major target of the STN neurons. We also reported that the most axonal boutons of STN neurons formed synaptic contacts with dendritic shafts containing mitochondria in the neostriatum, suggesting that the STN axons may tend to avoid dendritic spines, which lack mitochondria. The neostriatum contains various neuron types, which are divided into spiny projection neurons (making up ~90% of neostriatal neurons) and aspiny interneurons (smaller fractions). Given the modest number of boutons on relative straight and poorly branched axons that the STN neurons sent to the neostriatum, we hypothesized that the STN neurons might selectively innervate aspiny interneurons in the neostriatum. We addressed this issue by the triple immunofluorescence method. STN efferents were visualized with a Sindbis viral vector expressing palmitoylation site-attached green fluorescent protein (palGFP). The post-synaptic interneurons in the neostriatum, which are composed of cholinergic, parvalbumin-producing, nitric oxide synthase-expressing and calretinin-producing neuron types, were detected by immunofluorescence for their neurochemical markers. In addition, pre-synaptic boutons were labeled with vesicular glutamate transporter 2 (VGLUT2) immunoreactivity. The palGFP- and VGLUT2-immunoreactive boutons that were apposed to each type of post-synaptic interneuron were counted with confocal microscopy and statistically analyzed. The vast majority of axonal boutons that arose from the STN neurons were apposed to the somatodendritic compartment of various types of interneurons in the neostriatum, suggesting that the excitatory efferents from the STN may inhibit the activity of projection neurons indirectly via the neostriatal interneurons.