Death from acute diarrheal diseases has been greatly reduced in the past 20 years but still kills 0.8 million children per year. Death from diarrhea has decreased in direct proportion to use of oral rehydration solutions . However, only ~33% of cases of acute diarrhea currently use ORS. Drug therapy is needed to reduce the duration and volume loss from diarrhea. Unfortunately, there are no medications available that offer safe treatment for most cases of acute diarrhea which are not further diagnosed as to etiology before therapy must be initiated. Failure of drug development is in the 90% range when development is based on use of animal and cell models with lack of efficacy and side effects in humans. It has been suggested that having human models on which to develop drug therapy might increase the success. Recent development of a procedure to propagate three-dimensional “mini-intestines” from isolated human intestinal Lgr5+ stem cells has provide the opening to use human small intestine and colon in physiologic, pathophysiologic, regenerative medicine, developmental biologic studies. These models were development by Hans Clevers and his associates at the Hubrecht Institute, Utrecht, Netherlands. We have adopted their methods to carry out studies of human duodenum, jejunum, and ileum made from intestinal biopsies and surgical resections specimens in patients shown not to have intestinal disease and to have normal biopsies. This preparation is called human enteroids and consists of Lgr5+ stem cells, Paneth cells and based on culture conditions also goblet cells, entero-endocrine cells but no mesenchyme or nerves. The enterocytes are intestinal segment specific with duodenum and jejunum but not ileum expressing SGLT1 and ileum but not proximal intestine expressing the Na bile acid transporter A similar preparation made from IPS cells contains mesenchyme and is not intestinal segment specific. The IPS based preparation is called organoids. The enteroids develop polarity expressing apical markers such as villin and basolateral markers such as Na-K-ATPase and e-cadherin and have tight junctions marked by ZO-1. They can made to differentiate by Wnt3a removal. When differentiated but not when undifferentiated they express by immunofluorescence apical NHE3, DRA and CFTR and basolateral membrane NKCC1 . In the differentiated state there is expression of Muc2. Structurally, while the enteroids have areas of clustered expression of NHE3 and DRA containing cells, these cells also express CFTR and no clear villus structures are present; although villus like structures have been obtained when murne organoids are grown under the capsule of the murine kidney, indicating that further differentiation can be achieved. The human enteroids were characterized for transport function. Differentiated duodenal and jejunal enteroids exhibit NHE3 activity determined by two photon microscopy/SNARF-4F and CFTR activity indicated by forskolin-induced swelling inhibited by the CFTR inhibitor-172. CFTR enteroids do not swell with forskolin. These enteroids were used to evaluate host-pathogen interactions. Forskolin and cholera toxin inhibit proximal intestinal enteroid NHE3 activity, with the effect of forskolin rapidly reversible. Rotavirus replicates in these adult human enteroids. The rotavirus causes mild changes in the brush border within 2 hours and inhibits NHE3. The rotavirus replicates in these enteroids producing NSP4 up to 96h post infection with visualization of nascent virus particles within intracellular vesicls by EM . These human enteroids are also a model for EHEC related diarrhea, undergoing actin cytoskeletal changes similar to what occurs in diarrhea caused by enterohemorrhagic E. coli, during which the bacterial protease EspP induces macropinocytosis and transcytosis of Shiga toxins 1 and 2 to allow the toxins to enter the systemic circulation. Human enteroids represent a model to understand transport physiology and its normal physiologic regulation as well as pathophysiology in pathogen related diarrheal diseases. This platform will be tested for drug development testing the hypothesis that normal human intestine will provide insights for drug development that can not be gained from animal intestine and other cell models.