Recently we produced pancreatic polypeptide transgenic (PPTG) mice and found that PP was overexpressed in pancreatic islets. The present study examines development of four islet hormones in PPTG mice at embryonic days (ED) 15, 17, and 19, and in adult animals. Adult PPTG mice showed massive aggregation of PP-positive cells and glucagon-positive cells seen at the central area of the islets. Confocal laser microscopic study showed that three islet hormones (insulin, glucagon and PP) were completely overlapped in islets of PPTG mice. Overlapping of somatostatin/glucagon and somatostatin/PP were also increased at the peripheral area of the islets in adult PPTG mice compared to wild-type mice. In prenatal development of pancreatic islets of PPTG mice, somatostatin/glucagon overlapping cells appeared at ED 15, two days earlier than in wild-type mice. Differentiation of these somatostatin/glucagon double-positive cells into single-positive cells was disturbed in the PPTG mice during perinatal to postnatal periods. Differentiation of glucagon/insulin-double positive cells into single-positive cells was disturbed remarkably in postnatal development of the islets of PPTG mice. The present results suggest that early and overexpression of PP may engender the early appearance of somatostatin producing cells; however, that may disturb differentiation of multihormonal immature endocrine cells into single hormonal mature endocrine cells.