Close intra-arterial infusion of the appetite regulating peptide orexin-A stimulates bicarbonate secretion from the duodenal mucosa. The aim of the present study was to elucidate the ability of orexin-A to induce intracellular calcium signaling in acutely isolated duodenal enterocytes. Methods. Freshly isolated clusters of enterocytes, obtained from rat duodenal mucosa or human duodenal biopsies were loaded with fura-2 AM and mounted in a perfusion chamber. Crypt-like enterocytes were selected (caged) and changes in intracellular calcium concentration ([Ca²⁺]_i) were evaluated by fluorescence imaging. Total RNA was extracted from pellets of enterocytes, reverse transcripted to cDNA and expression of orexin receptors 1 and 2 (OX₁R and OX₂R), measured by quantitative real-time PCR. Results. Orexin-A at all concentrations tested (1-100 nM) increased [Ca²⁺]_i in enterocytes isolated from continuously fed rats, and the OX₁R-antagonist SB-334867 (10 nM) attenuated the response. The primary [Ca²⁺]_i response was a slow increase to a sustained plateau persisting after orexin-A removal, and a similar response was observed in enterocytes from human biopsies. In contrast to orexin-A, the OX₂R agonist (Ala¹¹, D-Leu¹⁵)-orexin-B (1-10 nM) did not induce calcium signaling. There were no significant [Ca²⁺]_i responses in enterocytes from animals food deprived overnight, and overnight fasting decreased (P < 0.01) enterocyte OX₁R as well as OX₂R mRNA. Conclusions. Induction of intracellular calcium signaling in isolated duodenal enterocytes is mediated primarily by OX₁R receptors. Short (overnight) food deprivation markedly depresses receptor expression and inhibits orexin-A induced increases in [Ca²⁺]_i. Studies of enterocyte signaling and intestinal secretion requires particular evaluation regarding feeding status.