Our previous findings indicate that lesions in the medial division of the medial geniculate nucleus (mMGN) prevent the acquisition of differential conditioning of bradycardia to acoustic stimuli in rabbits. In the present experiment, the effect of lesions in mMGN on retention of differential bradycardiac conditioning was examined. In addition, the possible involvement of auditory cortex in differential conditioning was investigated. Electrodes were chronically implanted in mMGN, the ventral division of the medial geniculate nucleus (vMGN), or auditory cortex. After 7 days of recovery, animals received one differential Pavlovian conditioning session. At the end of the session, lesions were produced through the implanted electrodes. All animals demonstrated differential bradycardiac conditioning during the prelesion session. Animals with vMGN lesions also demonstrated differential conditioning during the postlesion session. However, mMGN and auditory cortex lesion animals failed to demonstrate differential conditioning during the postlesion session due to an increased response magnitude to the unpaired tone (CS-). These data support the hypothesis that mMGN plays a role in differential conditioning of bradycardia to tonal stimuli. In addition, these findings suggest that a possible corticothalamic pathway may be involved in the inhibition of the response to the CS-.