K-cells are a sub-population of enteroendocrine cells that secrete GIP- a hormone that promotes glucose homeostasis and obesity. Therefore, it is important to understand how GIP secretion is regulated. GIP-producing (GIP/Ins) cell lines secreted hormones in response to many GIP secretagogues except glucose. In contrast, glyceraldehyde and methyl-pyruvate stimulated hormone release. Measurements of intracellular G-6-P, F-1,6-P2, and pyruvate levels, as well as glycolytic flux, in glucose-stimulated GIP/Ins cells indicated that glycolysis was not impaired. Analogous results were obtained using glucose-responsive MIN6 insulinoma cells. Citrate levels increased similarly in glucose-treated MIN6 and GIP/Ins cells. Thus, pyruvate entered the Krebs cycle. Glucose and methyl pyruvate stimulated 1.4- and 1.6-fold increases, respectively, in the ATP/ADP ratio in GIP/Ins cells. Glyceraldehyde profoundly reduced, rather than increased, ATP/ADP. Thus, nutrient-regulated secretion is K_ATP channel-independent. Antibody staining of mouse intestine demonstrated that enteroendocrine cells producing GIP, GLP-1, cholecystokinin, or somatostatin do not express detectable levels of Kir 6.1 or Kir 6.2 indicating that release of these hormones in vivo may also be K_ATP channel independent. Conversely, nearly all cells expressing chromogranin A or Substance P, and ~50% of the cells expressing secretin or serotonin, exhibited Kir 6.2 staining. Compounds that activate calcium mobilization were potent secretagogues for GIP/Ins cells. Secretion was only partially inhibited by verapamil suggesting calcium mobilization from intracellular as well as extracellular sources, independent from K_ATP channels, regulates secretion from some, but not all, sub-populations of enteroendocrine cells.