Background & aim:Some appetite regulating hormones including orexin-A and orexin-B are expressed in endocrine cells in intestinal mucosa as well as in the hypothalamus. We have compared effects of orexin-A on intracellular calcium [Ca²⁺]_i signaling in enterocytes acutely isolated from duodenum in continuously fed or food deprived rats. Methods: Rats had access to their regular food supply, or were deprived of food overnight. They were sacrificed at 8 am and the duodenal mucosa was harvested and mildly digested (collagenase/dispase) to yield clusters (10–100 cells) of duodenal enterocytes. Clusters were loaded with fura-2, mounted in a perfusion chamber and [Ca²⁺]_i was measured with fluororescence imaging. Results: Orexin-A (1–100 nM) in a dose-dependent manner induced [Ca²⁺]_i signaling in duodenal enterocytes isolated from continuously fed animals. The response was similar to those observed with carbachol or cholecystokinin. In contrast, there were no responses to orexin-A in enterocytes from overnight food deprived animals. The orexin (OX1) receptor antagonist SB-334867 was perfused at concentrations of 10 or 100 nM. The higher concentration of SB-334867 alone induced [Ca²⁺]_i signaling, similar to that observed with orexin-A, suggesting a partial agonist action. At the lower concentration of 10 nM, SB-334867 inhibited the response to 10 nM orexin-A but not that to 100 nM orexin-A. Conclusion: The orexogenic peptide orexin-A induces [Ca²⁺]_i signaling in duodenal enterocytes from fed animals. Overnight fasting abolishes the response. These results are in line with previous findings that short fasting abolishes the duodenal secretory response to orexin-A and thus strongly support that enterocyte OX1 receptors are involved in regulating intestinal secretion.