The protein nucleobindin-2 (NUCB2, also known as nesfatin) was recently implicated as a mediator of anorexia and catabolism in the central nervous system, and has been suggested to act as a cleaved and secreted messenger. Given the overlap of signalling molecules between the brain and pancreas, we have explored the presence of NUCB2 in the islets of Langerhans. We also performed an investigation of the dynamic regulation of pancreatic NUCB2 in different metabolic states. NUCB2-like immunoreactivity was detected by immunofluorescence in all human and rat islet beta-cells (as detected by co-localization with insulin), but not in other islet cells or in the exocrine pancreas. Islet NUCB2 content, as measured by enzyme immunoassay, did not change significantly following an overnight fast, but was substantially lower in islets isolated from an animal model of type 2 diabetes, the Goto-Kakizaki (GK) rats (48% of non-diabetic Wistar rat control). Serum levels, however, were not different between Wistar and GK rats. The release of NUCB2 from isolated rat islets was significantly elevated following glucose challenge (123%), but this effect was substantially lower than that observed for insulin (816%). In contrast, serum levels of NUCB2 showed a reversible decrease in an i.p. glucose tolerance test. These data suggest a role for NUCB2 in beta-cell function and a potential involvement in diabetic pathology. However, our findings, together with previous reports, appear more compatible with intracellular actions rather than with endocrine/paracrine communication, and suggest that NUCB2 in serum derives primarily from non-islet sources.