Enteroendocrine cells (EEC) secrete key gastrointestinal (GI) hormones such as gastric-inhibitory peptide (GIP), cholecystokinin (CCK) and ghrelin that are involved in nutrient sensing pathways and are often implicated in GI pathology. Traditionally, these cells have been classified by the ‘Wiesbaden’ system according to the specific hormone they contain. The enteroendocrine ‘I-cell’ is concentrated in the proximal duodenum and, according to this classification, secretes CCK, which acts to co-ordinate optimal digestion and absorption of nutrients and induce a satiety response to control food intake. The aim of this study was to determine the hormonal profile of duodenal I-cells. I-cells were purified by fluorescence activated cell sorting (FACS) of duodenal cells isolated from (CCK_eGFP)BJ203G transgenic mice (CCK-eGFP mice). Total RNA was prepared from eGFP+ (I-cells) and eGFP- cells, and subjected to semi-quantitative RT-PCR. RT-PCR showed enrichment for CCK, ghrelin, peptide YY (PYY), GIP, neurotensin, secretin, somatostatin and glucagon-like peptide-1 (GLP-1). Paraffin sections were prepared from the mid-duodenum segment of CCK-eGFP mice. Sections were subjected to immunostaining with antisera against proCCK, ghrelin, PYY, GIP and GLP-1. Initial studies showed 89% (±1.25 n=19) eGFP cells labelled positively for CCK, confirming the validity of the mouse model. Significant numbers of eGFP cells were shown to co-express ghrelin (50% ±1.71 n=25) or PYY (45% ±2.76 n=9) or GIP (36% ±1.85 n=10) or GLP-1 (14% ±2.14 n=10). Co-expression was confirmed by dual immunostaining that showed proCCK to be expressed alongside ghrelin, PYY, GIP or glucagon. This showed that CCK I-cells contain a number of key GI hormones. Analysis of high magnification micrographs revealed a complex pattern of vesicular staining. In dual labelling experiments some vesicles stained for only one hormone whereas others were positive for both hormones. Therefore, some hormones were co-packaged within a single vesicle, suggesting that I-cells have the ability to co-release CCK alongside other hormones. It is clear that I-cells have a complex repertoire of key gut hormones; thus EEC’s can no longer be simply classified as containing only one hormone and also suggests that further I-cell ‘subsets’ possibly exist. These data, combined with previous data showing the spectrum of nutrient-sensing receptors expressed in I-cells, suggest I-cells integrate several nutrient signals and have the ability to secrete a cocktail of hormones.