Orexins are hypothalamic peptides involved in the regulation of feeding, sleep and autonomic functions. Yet, orexins are also detected outside the CNS in peripheral organs like testis, kidney, adrenal, intestinal neuroendocrine cells and pancreatic islets. The effect of orexin on glucose induced insulin secretion is quite unclear. Orexin-1 receptor (OX1R) expression in rat insulinoma b-cell line INS-1E was confirmed by RT-PCR. Glucose stimulated (30min) insulin secretion was measured in the presence and absence of orexin-A (Ox-A, 20 min preincubation). [Ca2+]_i was measured using fura-2. Membrane depolarization was studied in current-clamp mode using standard perforated patch. Ox-A caused a significant and dose-dependent inhibition of glucose stimulated insulin secretion. At 5,5 mM glucose, 100 nM Ox-A reduced insulin secretion by app. 30%, whereas 500 nM Ox-A significantly inhibited insulin secretion (48%). At 16,7 mM glucose, insulin secretion was significantly inhibited by 100 nM Ox-A (33%) and 500 nM Ox-A (44%).100 nM Ox-A significantly reduced the elevation of [Ca2+]_i in response to glucose in PTX-insensitive manner. Ox-A (50, 100 or 100 nM) did not have any effect on forskolin stimulated cAMP production. In patch clamp studies, 100 nM Ox-A caused a 74 ± 12% reduction of glucose stimulated depolarization. Conclusions: Our results suggest that Ox-A has an inhibitory effect on insulin secretion in INS-1E cells via a decrease in intracellular calcium. Ox-A might thus participate in the regulation of insulin secretion in a paracrine manner inside the islets. Supported by the Academy of Finland and the Novo Nordisk