Protein intake robustly stimulates the secretion of the incretin hormone, glucagon- like peptide-1 (GLP-1) but the molecular mechanisms involved are not well understood. In particular, it is unknown whether proteins stimulate secretion by activation of luminal or basolateral sensors. We characterized the mechanisms using a physiologically relevant model-the isolated perfused proximal rat small intestine. Intraluminal protein hydrolysates derived from meat (peptone; 50 mg/mL) increased GLP-1 secretion 2.3-fold (from a basal secretion of 110 +/- 28 fmol/min). The sensory mechanisms underlying the response depended on di/tripeptide uptake through Peptide Transporter 1 (PepT1) and subsequent basolateral activation of the amino acid sensing receptor, Calcium-Sensing Receptor (CaSR), since inhibition of PepT1 as well as CaSR both attenuated the peptone-induced GLP-1 response. Supporting this, intraluminal peptones were absorbed efficiently by the perfused intestine (resulting in increased amino acid concentrations in the venous effluent) and infusion of amino acids robustly stimulated GLP-1 secretion. Inhibitors of voltage-gated L-type Ca2+ channels had no effect on secretion suggesting that peptone-mediated GLP-1 secretion is not mediated by L-cell depolarization with subsequent opening of these channels. Specific targeting of CaSR could serve as a target to stimulate the endogenous secretion of GLP-1.