The processing of preproghrelin in the stomach by prohormone convertase (PC) 1/3 produces ghrelin and possibly obestatin. In the neonate, the pancreas is also a major source of ghrelin. We compared the ontogeny of preproghrelin, ghrelin, obestatin, and PCs in the stomach and pancreas from rat embryos (day 21) and neonates (days 1, 6, 13, 21, and 28) by immunohistochemistry. In stomach, preproghrelin positive cells were present from embryonic day 21 and were in excess of ghrelin cells. The number of ghrelin positive cells progressively increased with age. When preproghrelin cells were immunoreactive for ghrelin, they were also immunoreactive for obestatin and PC1/3. In pancreas, we only found 0 to 2 preproghrelin positive cells per islet and each of these cells was also positive for ghrelin and obestatin. None of the ghrelin positive cells stained for insulin, but we observed ghrelin positive/glucagon negative and ghrelin positive/glucagon positive cells. Ghrelin positive cells contained PC1/3 or PC2. In summary, in stomach, an excess of preproghrelin positive cells compared with ghrelin/PC1/3 positive cells suggests that PC1/3 determines preproghrelin processing to ghrelin. In pancreas, the colocalization of PC1/3 or PC2 in ghrelin positive cells points to a role for both PCs in preproghrelin processing.