In order to assess the possible role of carboxypeptidase E (Cpe) in pro-CCK processing, tissues from the Cpe(fat)/Cpe(fat) mice were analyzed for CCK content and molecular forms using specific RIAs directed against different portions of the prohormone. Levels of amidated CCK were decreased by about 74% in whole brain of Cpe(fat)/Cpe(fat) mice in comparison to control mice, while levels of amidated CCK in intestine were only reduced by about 36%. In contrast, using an antiserum specific for CCK Gly Arg Arg, Cpe(fat)/Cpe(fat) mice brain had about 13-fold higher levels of this peptide relative to controls, while levels were identical in mutant and control duodenal tissue. This study demonstrates a regional difference in the involvement of Cpe in pro-CCK processing. The accumulation of CCK Gly Arg Arg in Cpe(fat)/Cpe(fat) brains provides definitive proof that the dibasic cleavage of the carboxyl terminus of pro CCK occurs on the carboxyl terminal of the dibasic, between the Arg and Ser as well as confirming that amidated CCK 8 in brain originates from CCK 8 Gly Arg Arg rather than from larger amidated peptides like CCK 22 or CCK 33. The Cpe(fat)/Cpe(fat) mouse phenotype obviously involves multiple endocrine defects, however, it is tempting to speculate that this severe CNS deficiency in CCK 8 may be related to the adult-onset obesity seen in this mutant mouse.