Neogenesis of β-cells is the process that allows the endocrine pancreas to meet the increasing demands in insulin secretion occurring with pregnancy and obesity, or following partial pancreatectomy. The critical question concerns the origin of insulin secreting cells. First, we determined the origin of pancreatic cells in a transgenic mouse model in which the tamoxifen-inducible MerCreMer protein was targeted to the Kit locus. These mice were cross-bred with an R-GFP reporter line to irreversibly label with GFP any cell that expressed c-kit at the time of tamoxifen administration. After tamoxifen for 6-8 months, acinar cells were intensely positive for GFP. Additionally, GFP-labeled β-cells were consistently found within the islets of Langerhans. Another fate mapping model was tested, the Kit mouse, in which a different region of the c-kit promoter was targeted. Following tamoxifen for 2-3 months, large regions of the mouse pancreas were positive for GFP, amylase and insulin, indicating that in both mice these specialized cells derived from c-kit-positive cells. For the detection of distinct intracellular proteins, a group of mice was treated with tamoxifen for 6 weeks; this protocol required tissue digestion, fixation and immunolabeling of the isolated cells and the analysis of the various compartments by flow cytometry. The freshly isolated pancreas showed large areas positive for native GFP. Importantly, the pool size of freshly isolated non-fixed cells showing native GFP was comparable to that present in fixed pancreatic cells displaying immunolabeled GFP. Within the entire compartment of pancreatic cells, the fraction of insulin, C-peptide and glucagon positive cells expressing GFP was 6%, 20% and 14%, respectively. The large percentage of non-endocrine cells expressing GFP may reflect the pool of acinar cells synthesizing amylase. Subsequently, the frequency of insulin-positive, C-peptide-positive and glucagon-positive cells labeled by GFP within each cell class was measured. The fraction of double positive cells averaged approximately 20% in all cases. In conclusion, these results strongly suggest that the mammalian pancreas contains a pool of c-kit-positive stem cells which are multipotent and may regulate organ homeostasis.