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Ruminal uptake of anions such as chloride and bicarbonate is essential for the maintenance of ruminal homeostasis in the ovine and bovine species and may involve a basolateral electrogenic efflux mechanism. In the current study, a previously identified chloride conductance of isolated cells of the ruminal epithelium (Am J Physiol Gastrointest Liver Physiol 289: 3, G508-20, 2005) was studied in more detail using the patch clamp technique. In the whole cell configuration of cells filled with (buffered) K-gluconate or Na-gluconate solutions, outward current could be reduced significantly (<70%; p < 0.001) by replacement of 130 mM of external chloride with gluconate. Similar effects were seen when chloride influx was blocked with DIDS (1mM). At 100 M, effects of DIDS and niflumic acid were significant, but small (≈10%, p < 0.05, n = 4 each). Nitrate (10 mM or 130 mM) had no effect on outward current. Replacement of chloride by HCO3- led to a 30% reduction in outward current, with some cells showing an increase in inward current. Excised inside out patch-clamp experiments in symmetrical and asymmetrical NMDG-anion solutions showed channels with a conductance of 350 ± 7 pS for chloride. We conclude that isolated cells of the ruminal epithelium express an anion channel with p(Cl-) ≈ p(NO3-) > p(HCO3-) > p(Gluconate-). This channel may play a role in the basolateral efflux of anions from the rumen. (DFG STU