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Abstract: The third forestomach of ruminants, the omasum, is an organ with absorptive functions for water, sodium, bicarbonate and SCFA. Techniques for the isolation and cultivation of omasal epithelial cells are missing and explain the lack of knowledge about the underlying transport proteins. In the current study, we present a new technique for the isolation of omasal epithelial cells. Tissues were removed immediately after slaughter and incubated at 37 °C in a purpose-built, Ussing-Chamber style device in Ca-free PBS with 0.25% trypsin added mucosally. Isolation of the replicating cells of the stratum basale was performed via fractional trypsinization as established for cells of the ruminal epithelium. Immunohistochemically, both freshly isolated and passaged omasal cells showed staining for cytokeratin AE1. Patch clamp experiments demonstrate a DIDS sensitive conductance with permeability to chloride and acetate resembling the maxi-anion channel of the ruminal epithelium. We propose a role for this channel in the basolateral efflux of SCFA from the omasum.