Short-chain fatty acids are absorbed in considerable amounts from the rumen. During transit through the epithelial layer, they are intensively metabolised. Interaction between intraepithelial metabolism and absorption, however, is hardly understood. The present study therefore compared the transepithelial transport of the easily metabolised n-butyrate with that of the more metabolism-resistant iso-butyrate both under in vivo conditions (isolated and washed reticulorumen) and in vitro conditions (Ussing chamber). Under in vivo conditions, net absorption of n-butyrate was significantly higher than that of iso-butyrate. The in vitro experiments showed that the higher net flux of n-butyrate was solely due to a higher mucosal-to-serosal flux, whereas the serosal-to-mucosal flux of butyrate was independent from the isoform. Blocking intraepithelial ATP delivery by 2,4-dinitrophenol abolished the net flux of n-butyrate. The study indicates that metabolism and/or ATP availability stimulates n-butyrate net absorption. By this, the metabolic activity of the epithelium may have a regulatory influence on absorption of n-butyrate.