Secretin, a peptide hormone of the gastrointestinal system, has been implicated in the etiology of autism. Our laboratory previously demonstrated the expression of secretin and its receptors in specific central neurons, and found for the first time that secretin is neuroactive in the cerebellum. We showed that bath application of secretin facilitated the release of GABA from terminals of basket cells. In the present report, we further characterize the actions of secretin in the cerebellum and provide evidence for the endogenous release of secretin in this brain area. First, by means of whole-cell patch-clamp recordings from Purkinje neurons in acutely prepared rat brain slices, we discovered that the facilitatory action of secretin on inhibitory inputs is partly dependent on the release of endogenous glutamate. In the presence of CNQX, an AMPA/kainate receptor antagonist, the facilitatory effect of secretin on GABA release was significantly reduced. Consistent with this idea, application of AMPA facilitates GABA release from inhibitory terminals, in a CNQX-sensitive manner. These data indicate that a direct and an indirect pathway mediate the action of secretin. Secondly, secretin is also active at the synapse between Purkinje neuron terminals and deep nuclei neurons. Thus, in voltage-clamped deep nuclei neurons, bath application of a low concentration of secretin increased the frequency but not the amplitude of miniature inhibitory post-ynaptic currents (mIPSCs). Therefore, secretin may act on presynaptic secretin receptors on the terminals of Purkinje cells. These data explain the fact that Purkinje cells express secretin receptor mRNA but do not respond directly to secretin, and indicate that secretin could act at multiple levels in the cerebellar circuits. Finally, to show that secretin is released endogenously, blocks of freshly dissected cerebella were challenged with 40 mM of KCl, or KCl in the presence of tetrodotoxin (TTX) or cadmium. The amount of secretin released was measured by commercially available EIA kits. Incubation with KCl almost doubled the rate of secretin release. This KCl-induced release was sensitive to TTX and cadmium suggesting the involvement of voltage-gated sodium and calcium channels. On the whole, these data give further and more solid evidence for the role of secretin as a neuropeptide in the central nervous system, and provide the rationale to further explore the relationship between secretin, cerebellum, and autism.