The epithelial brush border Na/H exchanger NHE3 accounts for the majority of intestinal and renal Na absorption. In the small intestine it is highly regulated related to eating; under fasting conditions it is active, becomes quickly inhibited after eating and then it is stimulated in the post-prandial state. The inhibitory aspect of this regulation is duplicated as part of the pathophysiology of diarrhea. Both stimulation and inhibition of NHE3 involve large multi-protein signaling complexes that form on the NHE3 C-terminus, which makes up its 377aa long intracellular regulatory domain. The size of the NHE3 complexes are estimated as high as 1.7 mDa by density gradient centfiguation, with changes in signaling leading to changes in the size of the complexes and the associating proteins. Insights in the specific complexes include: Domain 1: Close to the NHE3 N- terminus: a) By homology with NHE1, calcineurin homologous protein binds to the beginning of the NHE3 C-terminus (NHE3 aa 474-500). A peptide mimicking this domain and presumably altering CHP binding stimulates NHE3 activity ( Babich, Vadnagara and Di Sole. FASEB J 27: 4646-4658, 2013). b) Just downstream there is an alpha-helical domain at which ezrin directly binds (aa 504-528). Ezrin binding is required for basal NHE3 activity (the PI 3-kinase dependent component) and for stimulation of NHE3 by exocytosis by agonists such as EGF and LPA. Ezrin binding here is a regulated process. On one side of this alpha-helix are three clustered Ser; two of these are phosphorylated under basal conditions by sequential activation of two kinase,s GSK-3B and Akt. Without these phosphorylations, ezrin does not bind to this domain of NHE3. Thus this signaling complex takes part in basal and stimulated NHE3 activity by regulating direct ezrin binding to NHE3. Domain 2: Middle of the NHE3 C-terminus: multiple proteins associate with NHE3 between aa 585-606. This part of the protein is involved with regulation of basal NHE3 activity controlling both stimulatory and inhibitory contributions to basal activity. The identified associating proteins include two kinases CK2 and CaMKII, NHERF 1,2,and 3; PLCγ, CaM and slightly further downstream IRBIT. CK2 stimulates basal NHE3 activity. CaM KII inhibits basal NHE3 activity. Both kinases phosphorylate NHE3 further downstream in the C-terminus, CK2 at 719 and CaMKII at three sites. There are two other examples of three phosphorylation sites that are clustered in the NHE3 C-teminus being required for regulation of NHE3 by a single kinase (cGKII, RSK as well as CaMKII). This domain of NHE3 is also involved in regulated NHE3 activity, particularly by agonists that mimick the decrease that occurs after eating. Inhibition of NHE3 by elevated intracellular Ca2+ requires CK2 phosphorylation of NHE3, presence of NHERF2 and NHERF3 but not the presence of NHERF1. The NHERF2 and NHERF3 dependence requires hetero-dimerization of NHERF2 and NHERF3. A peptide which mimicks this domain of NHE3 stimulates basal NHE3 activity at low concentrations in multiple cell types, including human enteroids, and should be considered as a model for drug development to treat diarrhea. Conclusions: NHE3 is one of the most highly regulated transport proteins. The regulation requires two large, multi-protein containing signaling complexes that form on its C-terminus. One complex in domain 1 is involved in setting basal NHE3 activity and is necessary for acute stimulation of NHE3. This is regulated by control of direct binding of NHE3 to ezrin. The second complex in domain 2 sets basal NHE3 activity by both increasing and decreasing NHE3 activity by altering its phosphorylation state. This complex takes part in inhibition of NHE3 activity which mimics the NHE3 inhibition that occurs immediately after eating, which spreads digestive enzymes over the absorptive/digestive surface of the intestine and represents the major function of NHE3 in digestion. Peptide mimicks of the parts of NHE3 which form the large signaling complexes stimulate NHE3 activity and thus identify NHE3 as a drug target for treating diarrhea.