Most peptide hormone genes are, in addition to endocrine cells, expressed also in neurons. The peptide hormone cholecystokinin (CCK) is expressed in different molecular forms in cerebral neurons and intestinal endocrine cells. In order to understand this difference, we have examined the roles of the neuroendocrine prohormone convertases, PC1/3, PC2 and PC5/6, by measurement of proCCK, processing intermediates and bioactive, -amidated and O-sulfated CCK peptides in cerebral and jejunal extracts of null mice, controls, and in the PC5/6-expressing SK-N-MC cell-line. In PC1/3 null mice, the synthesis of bioactive CCK peptide in the gut was reduced to 3% of the translational product, all of which was in the form of -amidated and tyrosine O-sulfated CCK-22, whereas the neuronal synthesis in the brain was largely unaffected. This is opposite to the PC2 null mice in which only the cerebral synthesis was affected. SK-N-MC cells, which do not express neither PC1/3 nor PC2, synthesized alone the processing intermediate, glycine-extended CCK-22. Immunocytochemistry confirmed that intestinal endocrine CCK-cells in wild-type mice express PC1/3, but no PC2. In contrast, cerebral CCK-neurons contain PC2 and only little, if any, PC1/3. Taken together, the data indicate that PC1/3 governs the endocrine and PC2 the neuronal processing of proCCK, whereas PC5/6 contributes only to a modest endocrine synthesis of CCK-22. The results suggest that the different peptide patterns in the brain and the gut are due to different expression of prohormone convertases.