Objective: The third forestomach of ruminants, the omasum, is an organ of singular anatomy which prepares fermented ingesta for acidification in the glandular stomach via poorly understood mechanisms. In vivo and in vitro studies support a considerable role in the uptake of short chain fatty acids (SCFA), with apical influx possibly occurring in the protonated form, while the route for basolateral efflux is obscure. Progress in understanding the underlying mechanisms is hindered by the lack of a cell culture model. Methods: Tissues were removed immediately after slaughter and incubated in a custom-made, Ussing-Chamber style device. Apical cell layers were removed by fractional trypsinization, allowing isolation and cultivation of the replicating cells of the stratum basale. Cultured cells were studied using immunohistochemistry and patch clamp techniques. Results: Immunohistochemical staining showed clear tight junctional staining for claudin-1, -4 and -7 as well as for occludin. Whole cell patch clamp experiments revealed DIDS sensitive conductances for Cl^- and acetate^- with p(Cl) > p(acetate). Permeability to propionate^- was demonstrated using the inside out configuration of the patch clamp technique, where large single channel events could be observed, the size of which changed with the potential and the anions present. Data could be fitted with a model assuming independence, yielding conductances of ~ 350 pS for chloride, ~ 100 pS for propionate, and < 40 pS for gluconate. Conclusions: We introduce a method allowing the isolation of cells from the omasal epithelium. Cultured cells were shown to express tight junction proteins specific to cells of epithelial origin and important for maintaining paracellular barrier function. The cells were also shown to express a large conductance anion channel resembling that found in the rumen. We propose a role for this channel in the basolateral efflux of propionate and acetate from the omasum.