During posttranslational processing to generate CCK 8, pro-cholecystokinin (CCK) undergoes endoproteolytic cleavage at three sites. Several studies using endocrine and neuronal tumor cells in culture and recombinant enzymes and synthetic substrates in vitro have pointed to the subtilisin/kexin-like enzymes prohormone convertase (PC) 1, PC2, and PC5 as potential candidates for these endoproteolytic cleavages. In these experimental models, they all appear to be able to cleave pro-CCK to make the correct products. One rodent model has provided information about the role of PC2. PC2 knockout mouse brains had less CCK 8 than wild-type, although a substantial amount of CCK was still present. The degree to which CCK levels were reduced in these mice was regionally specific. These data indicated that PC2 is important for normal production of CCK but that it is not the only endoprotease that is involved in CCK processing. To evaluate whether PC1 and PC5 are possible candidates for the other enzymes involved in CCK processing, the distribution of PC1, PC2, and PC5 mRNA was studied in rat brain. Their colocalization with CCK mRNA was examined using double-label in situ hybridization. PC2 was the most abundant of these enzymes in terms of the intensity and number of cells labeled. It was widely colocalized with CCK. PC1 and PC5 mRNA-positive cells were less abundant, but they were also widely distributed and strongly colocalized with CCK in the cerebral cortex, hippocampus, amygdala, ventral tegmental area, and substantia nigra zona compacta. The degree of colocalization of the enzymes with CCK was regionally specific. It is clear that PC1 and PC5 are extensively colocalized with CCK and could be participating in CCK processing in the rat brain and may be able to substitute for PC2 in its absence. These three enzymes may represent a redundant system to ensure production of biologically active CCK.