Intact cerebellar structures (i.e., deep nuclei and perhaps cortex) are essential for acquisition of both simple delay and trace eyeblink (EB) conditioning. However, successful trace conditioning also requires intact cortico-limbic structures (i.e., hippocampus, medial thalamus, and medial prefrontal cortex, mPFC). A direct connection between the cerebellum and ventrolateral thalamic nuclei (VLTN) has been demonstrated in several species. Since VLTN projects to both premotor and prefrontal cortex, it may be an essential link in a cerebellar-thalamic-prefrontal circuit that provides the CNS substrate for acquisition of the trace EB CR. The current studies thus assessed the role of the VLTN on trace EB conditioning in New Zealand albino rabbits. We first verified afferent connections to the mPFC (Brodmann's area 32) from the VLTN, by injecting the retrograde tracer Flourogold(c) into area 32. Strong labeling in VLTN from terminal projections to mPFC were found. We next assessed the role of VLTN in trace eyeblink conditioning in animals that received either sham or ibotenic acid VLTN lesions. EB conditioning began with 10 consecutive daily sessions of trace conditioning, followed immediately by 4 days of extinction, and then 4 days of delay conditioning. VLTN lesions significantly impaired acquisition of both trace and delay conditioning, and impaired extinction. These findings, thus confirm the importance of the VLTN in a postulated cerebellar-thalamic-prefrontal circuit that underlies successful trace, as well as delay EB conditioning.