Degradation of Boc-CCK27-33 [Boc-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-PheNH2) a fully potent analog of CCK8 was studied on synaptic plasma membranes from pig brain cortex. Characterization of the metabolites was performed by HPLC. This allowed to show the hydrolysis of the Asp-Phe bond by the neutral endopeptidase "enkephalinase", and the cleavages at the Met-Gly and Trp-Met bonds by PCMB sensitive enzymes. Similar results were observed using Boc(diNle28,31)-CCK27-33 as substrate. To investigate the biological relevance of these enzymes in the CCK8 metabolism, the degradation studies were performed on rat brain slices, with [3H]Boc(diNle28,31)CCK27-33 as substrate. Using these more physiological preparations i.e. striatal or cortex slices, the tritiated probe was cleaved at the Nle-Gly and Gly-Trp bonds. These degradation pathways were almost completely inhibited by PCMB, but in the striatum this inhibition process induced the appearance of a small peak corresponding to the action of enkephalinase. Taken together these results seem to indicate that thiol proteases play a crucial role in the CCK8 metabolism but that enkephalinase is virtually not involved.