Enteroendocrine (EE) cells represent complex, rare, and diffusely-distributed intestinal epithelial cells making them difficult to study in vivo. A specific sub-population of EE cells called Gut K-cells produces and secretes glucose-dependent insulinotropic peptide (GIP), a hormone important for glucose homeostasis. The factors that regulate hormone production and secretion, as well as the timing of peptide release, are remarkably similar for K-cells and islet beta-cells suggesting engineering insulin production by K-cells is a potential gene therapeutic strategy to treat diabetes. K-cell lines could be used to study the feasibility of this potential therapy and to understand Gut K-cell physiology in general. Heterogeneous STC-1 cells were transfected with a plasmid (pGIP/Neo) encoding neomycin phosphotransferase, driven by the GIP promoter-only cells in which the GIP promoter was active survived genetic selection. Additional clones expressing pGIP/Neo plus a GIP promoter/insulin transgene were isolated-only doubly transfected cells produced preproinsulin mRNA. Bioactive insulin was stored and then released following stimulation with arginine, peptones, and bombesin-physiological GIP secretagogues. Like K-cells in vivo, the GIP/insulin-producing cells express the critical glucose sensing enzyme, glucokinase. However, glucose did not regulate insulin or GIP secretion or mRNA levels. Conversely, glyceraldehyde and methyl-pyruvate were secretagogues, indicating cells depolarized in response to changes in intracellular metabolite levels. Potassium channel opening drugs and sulphonylureas had little effect on insulin secretion by K-cells. The K-cell lines also express relatively low levels of Kir 6.1, Kir 6.2, SUR1, and SUR2 suggesting secretion is independent of K(ATP) channels. These results provided unexpected insights into K-cell physiology and our experimental strategy could be easily modified to isolate/characterize additional EE cell populations.