β-cell neogenesis from ductal precursors, and possibly from other pancreatic cell types, contributes to the expansion of β-cell mass during development and after diabetogenic insults in rodents. Using a mathematical model−based analysis of β-cell mass, replication, and size, we recently demonstrated that neogenesis is also quantitatively important to the expansion of β-cell mass during prolonged hyperglycemia. In the present study, we examined the morphological appearance of neogenic focal areas, duct cell replication, and β-cell cluster size distribution in male Sprague Dawley rats infused with either saline or 50% glucose (2 ml/h) for 0, 1, 2, 3, 4, 5, or 6 days. Pancreatic tissue characterized by a high density of small duct-like structures, previously described as neogenic focal areas, were present in glucose-infused rats after 2, 3, or 4 days of infusion. The cross-sectional area of the pancreas characterized as focal tissue peaked after 3 days of infusion at 2.9 ± 0.8%. In contrast to the partial pancreatectomy model of β-cell regeneration, duct cell replication was not increased before or during focal area formation. However, the replication rate of cells in the duct-like structures of the focal areas was twofold greater than in cells of the common pancreatic duct and 15- to 40-fold greater than in cells of small, medium, and large ducts. Duct-cell replication was significantly reduced in small, medium, and large ducts of glucose as compared to saline-infused rats (0.21 ± 0.02 vs. 0.48 ± 0.04%; P < 0.03). Duct-associated β-cell mass was not different in glucose- and saline-infused rats (P = 0.78), whereas the number of acinar-associated single β -cells increased by 70% after 3 and 4 days of glucose infusion. In addition to small duct-like structures, focal areas had considerable T-cell infiltration (151 ± 30 T-cells/ mm²). There was also an increase in T-cell infiltration in acinar tissue of glucose as compared to saline-infused rats (0.43 ± 0.11 vs. 0.03 ± 0. 01 T-cells/mm²; P < 0.0001). In conclusion, these data suggest that neogenic focal areas in these glucose-infused rats do not arise from replication and differentiation of ductal progenitor cells. Rather, acinar cell transdifferentiation into β-cells and acinar cell dedifferentiation into neogenic focal areas lead to new β-cell formation during prolonged hyperglycemia.