The opiate peptide enkephalin has been immunohistochemically localized within specific populations of climbing fibers and mossy fibers in the opossum's cerebellum. The intention of the present study was to determine the physiological effects of this peptide on Purkinje cell activity as well as to examine interactions between this peptide and the excitatory amino acids glutamate and aspartate. Iontophoretic application of enkephalin onto Purkinje cells in the posterior lobe vermis and adjacent hemisphere suppressed activity in nine of 16 (56%) spontaneously active units. Enkephalin increased the spontaneous activity of one unit and had no effect on six (38%) of the units. In addition, this peptide blocked the excitatory effects elicited by iontophoretic application of glutamate in 34 of 35 (97%) units tested and of aspartate in all cases. Enkephalin had no effect on one cell activated by glutamate. Simultaneous application of naloxone, a nonspecific opiate receptor antagonist, did not block the suppressive effects of enkephalin. Rather, there was a potentiation of suppressive responses as compared to that seen when each is applied alone. The results suggest that classically defined excitatory afferent projections to the cerebellum may be capable of both exciting and suppressing the activity of their target neurons. The excitatory action is likely mediated by an amino acid, whereas the release of the peptide enkephalin results in a decrease in unit activity. Further, it appears that enkephalin mediates its suppressive effect through mechanisms that are not mediated by opioid receptors.