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The transport of peptides was studied with isolated preparations of rumen and omasum tissue of sheep by using the conventional Ussing-chamber method and isolated ruminal cells (REC). Mucosal addition of glycyl-L-glutamine, captopril (angiotensin converting enzyme inhibitor) or cefadroxil (beta-lactam antibiotic) did not change the short-circuit current (I(sc)), or tissue conductance (G(t)). The intracellular pH, pH(i), in isolated REC was not influenced by the addition of peptides to the buffer solution. These findings do not support the assumption of proton-coupled or electrogenic peptide transport. The determination of unidirectional flux rates of the peptide D-phenylalanyl-L-alanine (2,3-(3)H) showed that the flux rate in the serosal-mucosal direction, J(sm), was greater than J(ms), leading to a small net secretion of peptide. Transport was not significantly inhibited by the serosal addition of ouabain. Enhancing the paracelluIar permeability by an increase of osmotic pressure in the mucosal solution (FREYER and MARTENS, Proc. Soc. Nutr. Physiol. 8, 80, 1999) caused an increase of G(t) and significantly higher transport rates of peptide. The flux rates of peptides (in the nanomolar range) may therefore represent passive and possibly paracellular diffusion and are not of nutritional importance.