The nictitating membrane/eyelid responses of 18 rabbits were classically conditioned using cerebellar mossy-fiber stimulation as a conditioned stimulus (CS) and air puff as an unconditioned stimulus (US). The dorsolateral, lateral, and medial pontine nuclei and the middle cerebellar peduncle were effective stimulation-CS sites for training. In one group of rabbits, robust conditioned eyelid responses were produced with paired trials and subsequently extinguished with CS-alone and explicitly unpaired presentation of the CS and US. In a second group of rabbits, no conditioned responses were evident for 4 days of unpaired CS and US presentations. Conditioned responses did develop, however, after paired training was begun. Lesions of the interpositus nucleus of the cerebellum completely abolished the conditioned responses of a third group of rabbits overtrained with the mossy-fiber CS and air-puff US. These results support previous studies which have demonstrated that the cerebellum is critically involved in acquisition and retention of simple learned responses. In addition, the present results support previous theories of cerebellar function which have proposed that mossy fibers supply critical "learning" input to the cerebellum for acquisition and retention of motor skills.