One major unresolved question in the field of pancreas biology is whether ductal cells have the ability to generate insulin-producing β-cells. Conclusive examination of this question has been limited by the lack of appropriate tools to efficiently and specifically label ductal cells in vivo. We generated Sox9CreER^T2 mice, which, during adulthood, allow for labeling of an average of 70% of pancreatic ductal cells, including terminal duct/centroacinar cells. Fate-mapping studies of the Sox9⁺ domain revealed endocrine and acinar cell neogenesis from Sox9⁺ cells throughout embryogenesis. Very small numbers of non-β endocrine cells continue to arise from Sox9⁺ cells in early postnatal life, but no endocrine or acinar cell neogenesis from Sox9⁺ cells occurs during adulthood. In the adult pancreas, pancreatic injury by partial duct ligation (PDL) has been suggested to induce β-cell regeneration from a transient Ngn3⁺ endocrine progenitor cell population. Here, we identify ductal cells as a cell of origin for PDL-induced Ngn3⁺ cells, but fail to observe β-cell neogenesis from duct-derived cells. Therefore, although PDL leads to activation of Ngn3 expression in ducts, PDL does not induce appropriate cues to allow for completion of the entire β-cell neogenesis program. In conclusion, although endocrine cells arise from the Sox9⁺ ductal domain throughout embryogenesis and the early postnatal period, Sox9⁺ ductal cells of the adult pancreas no longer give rise to endocrine cells under both normal conditions and in response to PDL.