The trace conditioning paradigm in which the conditioned stimulus-unconditioned stimulus (CS-US) interval is longer and the US and CS do not overlap requires retention of a 'trace' of the CS to associate it with the US. This task is difficult, requiring for learning about 5 times the number of trials to criterion as the standard delay task. The present study was undertaken to determine if: the cerebellar interpositus nucleus is essential in trace as well as in delay conditioning; and the lateral cerebellar cortex is involved when CS-US association over longer time intervals is required. Sixteen adult male New Zealand white rabbits had recording and in some cases lesion electrodes surgically implanted before training. They were trained daily with 126 paired trials of tone CS and airpuff US. The trace period between CS offset and US onset was 500 ms. Mean number of trials to criterion of 8/9 CRs was 466.9 trials. After a day of overtraining the animals had one of 3 surgeries: electrolytic lesion of the left cerebellar interpositus nucleus (n = 3); aspiration of the left lateral cerebellar cortex (HVI and HVIIA) and underlying cerebellar nuclei (n = 4); or aspiration of HVI and HVIIA only (n = 9). Rabbits with only HVI and HVIIA removed exhibited a transient decrease in CRs but relearned with significant savings. The amplitude, area and latency of pre- and postlesion conditioned responses (CRs) was similar. When the interpositus nucleus was damaged, the animals' capacity for CRs on the side ipsilateral to the lesion was permanently abolished. It is concluded that the cerebellar interpositus nucleus but not the overlying lateral cerebellar cortex is essential for retention of trace classical conditioning. Nevertheless, the temporary abolition of CRs by removal of lateral cerebellar cortex suggests that the cerebellar cortex does play an important role in learning and retention of the trace conditioned response.