Unidirectional 22Na+ and 36Cl- fluxes were determined in short-circuited, stripped rumen mucosa from sheep by using the Ussing chamber technique. In both CO2/HCO3(-)-containing and CO2/HCO3(-)-free solutions, replacement of gluconate by short-chain fatty acids (SCFA, 39mM) significantly enhanced mucosal-to-serosal Na+ absorption without affecting the Cl- transport in the same direction. Short-chain fatty acid stimulation of Na+ transport was at least partly independent of Cl- and could almost completely be abolished by 1 mM mucosal amiloride, while stimulation of Na+ transport was enhanced by lowering the mucosal pH from 7.3 to 6.5. Similar to the SCFA action, raising the PCO2 in the mucosal bathing solution led to an increase in the amiloride-sensitive mucosal-to-serosal Na+ flux. Along with its effect on sodium transport, raising the PCO2 also stimulated chloride transport. The results are best explained by a model in which undissociated SCFA and/or CO2 permeate the cell membrane and produce a raise in intracellular H+ concentration. This stimulates an apical Na+/H+ exchange, leading to increased Na+ transport. The stimulatory effect of CO2 on Cl- transport is probably mediated by a Cl-/HCO3- exchange mechanism in the apical membrane. Binding of SCFA anions to that exchange as described for the rat distal colon (Binder and Mehta 1989) probably does not play a major role in the rumen.