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Electroneutral Na transport across rumen epithelium is mediated by a Na+/H+ exchange mechanisms which is inhibited by amiloride (> 100 μM). The underlying NHE isoform for transepithelial Na transport was characterized in vitro by application of the specific inhibitor HOE642 for NHE1 and S3226 for NHE3. S3226 (1 μM; NHE3 inhibitor) abolished electroneutral Na transport under control condition and also the SCFA induced increase of Na transport via NHE. However, HOE642 (30 μM; NHE1 inhibitor) did not change Na transport rates. It is concluded that the isoform NHE3 mediates electroneutral Na transport and not the isoform NHE1 as proposed by Graham et al. (Am. J. Physiol. 292: R997-1007, 2007).