CCK is a peptide hormone released predominantly from GI mucosal enteroendocrine I cells located in the proximal small intestine. CCK stimulates gallbladder contraction, exocrine pancreatic secretion, delayed gastric emptying and satiety. Physiologic studies in animals and humans show that CCK secretion is stimulated by dietary medium to long chain fatty acids, aromatic amino acids and a luminal CCK releasing peptide. However, the precise molecular basis for CCK cell sensing of luminal nutrients remains unclear. This is largely due to the relative rarity (<1% of mucosal cells) and highly dispersed distribution of enteroendocrine cells, such as CCK I cells, along the GI tract. The absence of a faithful cell culture model and the lack of a method for accumulating pure enteroendocrine cells in general and I cells in particular has hindered progress in determining the molecular basis of nutrient chemosensation in the GI tract. We utilized a BAC transgenic mouse with the enhanced green fluorescent protein (EGFP) gene inserted immediately upstream of the CCK coding sequence to identify CCK producing I cells. Immunohistochemistry of gastrointestinal frozen sections stained with rabbit anti-CCK antibody was used to assess the faithful expression of the transgene only in CCK producing I cells. Small mucosal intestine cells were dispersed using 1mM EDTA and collagenase to produce a population of single cells. Dispersed EGFP positive cells representing CCK producing I cells was isolated by fluorescent activated cell sorting (FACS). Total RNA extracted from the pure population of CCK-EGFP positive I cells was reversed transcribed and analyzed by Affymetrix mouse genome 430-2.0 GeneChip. This same CCK-EGFP positive I cell preparation was utilized to assess a variety of secretagogues for stimulated release of CCK subsequently measured by radioimmunoassay. Our studies show that EGFP is faithfully expressed in CCK expressing I cells located in the proximal small intestinal epithelium in CCK-EGFP BAC transgenic mouse. Pure (95%) CCK expressing I cells were successfully isolated from dispersed intestinal cells via FACS of EGFP expressing cells. The gene expression profile of CCK expressing cells analyzed by microarray studies suggested multiple candidate genes, such as GPR40, GPR120, and the calcium sensing receptor (CaR) that may act as luminal sensors for nutrient stimulated release of CCK. The presence of GPR40 and GPR120 transcript expression in CCK expressing cells was verified by qRT-PCR. Consistent with our microarray and qRT-PCR data, FACS isolated pure CCK expressing cells release CCK in response to the free fatty acid and phenylalanine in a time dependent manner. In vivo studies in WT and GPR40 KO mice gavaged with oleic acid support the role of GPR40 as a sensor for LCFA.