The role of aminopeptidase A (APA) in inactivating cholecystokinin (CCK-8) was investigated in in vitro and in vivo experiments. EC 33 (3-amino-4-thio-butyl sulfonate), a selective APA inhibitor, decreased the formation of CCK7 after incubation of CCK-8 with rat brain synaptic membranes. The Km of purified APA for CCK-8, determined by quantifying CCK-7 production, was 144 microM and the Kcat 1400 s-1 . EC 33 protected endogenous CCK-like immunoreactivity (CCK-LI) released from brain slices by evoked depolarizations. The serine/thiol protease inhibitor Ala-Ala-Pro-Val-COCH2Cl (AAPV), alone or in combination with EC 33, did not modify significantly the level of CCK-LI released from the hippocampus, whereas it weakly protected the CCK-LI released from the cortex. Intracerebroventricular coadministration of CCK-8 and EC 33 in mouse brain led to a significant increase in the apparent affinity of CCK-8 as determined by the inhibition of the selective CCKB receptor agonist binding [3H]pBC 264 (ID50 = 88 pmol vs. 8250 pmol for CCK-8 alone); AAPV was less potent (ID50 = 445 pmol). In the same experiment the ID50 of pCCK-8, protected from aminopeptidases by a propionyl group was 86 pmol. These results strongly suggest that APA plays a major role in the inactivating pathway of CCK-8.