The mediodorsal nucleus of the thalamus (MD) represents the main subcortical structure that projects to the prefrontal cortex (PFC) and it regulates key aspects of the cognitive functions of this region. Within the PFC, GABA local circuit neurons shape the activity patterns and hence the “memory fields” of pyramidal cells. Although the connections between the MD and PFC are well established, the ultrastructural relationships between projecting fibers from the MD and different subclasses of GABA cells in the PFC are not known. In order to address this issue in the rat, we examined MD axons labeled by tract-tracing in combination with immunogold-silver to identify different calcium-binding proteins localized within separate populations of interneurons. Electron micrographic examination of PFC sections from these animals revealed that MD terminals made primarily asymmetric synapses onto dendritic spines and less commonly onto dendritic shafts. Most of the dendrites receiving MD synaptic input were immunoreactive for parvalbumin (ParV), whereas MD synapses onto dendrites labeled for calretinin or calbindin were less frequent. We also observed that some MD terminals were themselves immunoreactive for calcium-binding proteins, again more commonly for ParV. These results suggest that the MD exerts a dual influence on PFC pyramidal cells: direct inputs onto spines and an indirect influence mediated via synapses onto each subclass of interneurons. The apparent preferential input to ParV cells endows MD afferents with a strong indirect inhibitory influence on pyramidal neuron activity by virtue of ParV cell synapses onto soma, proximal dendrites, and axon initial segments. J. Comp. Neurol. 490:220–238, 2005. © 2005 Wiley-Liss, Inc.