Monocarboxylate/H⁺ co-transporters such as MCT (SLC16A) have been suggested to mediate trans-ruminal fluxes of, short chain fatty acids, ketone bodies and lactate. Using an RT-PCR approach we demonstrate expression of both MCT1 (SLC16A1) and MCT2 (SLC16A7) mRNA in isolated bovine rumen epithelium. cDNA sequence from these PCR products combined with overlapping EST sequence data allowed compilation of the complete open reading frames for MCT1/2. Immunohistochemical localisation of MCT1 shows plasma-membrane staining in the stratum basale cells, with the basal aspects of the cells being intensely stained. There was decreasing immunostaining in the cell layers towards the rumen lumen, with weak staining in the stratum spinsoum. The stratum granulosum and the stratum corneum were essentially negative. Since MCT transport will acid load the cytosol of cells, the expression and location of Na⁺/H⁺ exchanger family members within rumen epithelium was determined. RT-PCR demonstrates expression of multiple family members, including NHE1-3 and NHE8. In contrast to MCT1, NHE1 was predominately localised to the stratum granulosum with a progressive decrease in immunostaining towards the stratum basale. NHE2 immunostaining was present mainly at an intracellular location in stratum basale, stratum spinosum and stratum granulosum cells . Given the anatomical localisation of MCT1, NHE1/2 the mechanism of trans-ruminal short chain fatty acids (SCFA), ketone body and lactate transfer is discussed in relation to a functional model of the rumen epithelium comprising an apical permeability barrier at the stratum granulosum with a cell syncitium linking the stratum granulosum to the blood-facing stratum basale.