The intestine secretes a range of hormones with important local and distant actions, including the control of insulin secretion and appetite. A number of enteroendocrine cell types have been described, each characterized by a distinct hormonal signature, such as K-cells producing glucose-dependent insulinotropic polypeptide (GIP), L-cells producing glucagon-like peptide-1 (GLP-1), and I-cells producing cholecystokinin (CCK). To evaluate similarities between L-, K-, and other enteroendocrine cells, primary murine L- and K-cells, and pancreatic α- and β-cells, were purified and analyzed by flow cytometry and microarray-based transcriptomics. By microarray expression profiling, L cells from the upper small intestinal (SI) more closely resembled upper SI K-cells than colonic L-cells. Upper SI L-cell populations expressed message for hormones classically localized to different enteroendocrine cell types, including GIP, CCK, secretin, and neurotensin. By immunostaining and fluorescence-activated cell sorting analysis, most colonic L-cells contained GLP-1 and PeptideYY In the upper SI, most L-cells contained CCK, approximately 10% were GIP positive, and about 20% were PeptideYY positive. Upper SI K-cells exhibited approximately 10% overlap with GLP-1 and 6% overlap with somatostatin. Enteroendocrine-specific transcription factors were identified from the microarrays, of which very few differed between the enteroendocrine cell populations. Etv1, Prox1, and Pax4 were significantly enriched in L-cells vs. K cells by quantitative RT-PCR. In summary, our data indicate a strong overlap between upper SI L-, K-, and I-cells and suggest they may rather comprise a single cell type, within which individual cells exhibit a hormonal spectrum that may reflect factors such as location along the intestine and exposure to dietary nutrients.