The auditory conditioned stimulus (CS) pathway that is necessary for delay eyeblink conditioning was investigated using reversible inactivation of the medial auditory thalamic nuclei (MATN) consisting of the medial division of the medial geniculate (MGm), suprageniculate (SG), and posterior intralaminar nucleus (PIN). Rats were given saline or muscimol infusions into the MATN contralateral to the trained eye before each of four conditioning sessions with an auditory CS. Rats were then given four additional sessions without infusions to assess savings from the initial training. All rats were then given a retention test with a muscimol infusion followed by a recovery session. Muscimol infusions through cannula placements within 0.5 mm of the MGm prevented acquisition of eyeblink conditioned responses (CRs) and also blocked CR retention. Cannula placements more than 0.5 mm from the MATN did not completely block CR acquisition and had a partial effect on CR retention. The primary and secondary effects of MATN inactivation were examined with 2-deoxy-glucose (2-DG) autoradiography. Differences in 2-DG uptake in the auditory thalamus were consistent with the cannula placements and behavioral results. Differences in 2-DG uptake were found between groups in the ipsilateral auditory cortex, basilar pontine nuclei, and inferior colliculus. Results from this experiment indicate that the MATN contralateral to the trained eye and its projection to the pontine nuclei are necessary for acquisition and retention of eyeblink CRs to an auditory CS.