The development of a culture of the normal mammalian jejunum motivated this work. Isolated crypt cells of the dog jejunum were induced to form primary cultures on Snapwell filters. Up to seven subcultures were studied under the electron microscope and in Ussing chambers. Epithelial markers were identified by rtPCR, western blot, and immunofluorescent staining. Confluent monolayers exhibit a dense apical brush border, basolateral membrane infoldings, desmosomes and tight junctions expressing ZO-1, occludin-1, and claudin-3 and -4. In OptiMEM medium fortified with epidermal growth factor, hydrocortisone and insulin, monolayer transepithelial voltage was -6.8 mV (apical side), transepithelial resistance was 1050 cm², and short-circuit current (I_sc) was 8.1 µA/cm². Transcellular and paracellular resistances were estimated as 14.8 kcm² and 1.1 kcm² respectively. Serosal ouabain reduced voltage and current towards zero, as did apical amiloride. The presence of mRNA of ENaC was confirmed. Na/D-glucose cotransport was identified with an antibody to SGLT1. The unidirectional mucosa to serosa Na⁺ flux (19 nmols^.min^-1.cm^-2) was twice as large as the reverse flux, and net transepithelial Na⁺ flux was nearly double the amiloride-sensitive short-circuit current. In plain Ringer solution, the amiloride-sensitive I_sc went towards zero. Under these conditions plus mucosal amiloride, serosal db-cAMP elicited a Cl^--dependent I_sc consistent with the stimulation of transepithelial Cl^- secretion. In conclusion, primary cultures and subcultures of the normal mammalian jejunum form polarized epithelial monolayers with 1) the properties of a leaky epithelium, 2) claudins specific to the jejunal tight junction, 3) transepithelial Na⁺ absorption mediated in part by SGLT1 and ENaC, and 4) electrogenic Cl^- secretion activated by cAMP.