High-producing ruminants are commonly fed large amounts of concentrate to meet their high energy demands for rapid growth or high milk production. However, this feeding strategy can severely impair rumen functioning, leading to subacute ruminal acidosis. Subacute ruminal acidosis might have consequences for electrophysiological properties by changing the net ion transfer and permeability of ruminal epithelia, which may increase the uptake of toxic compounds generated in the rumen into the systemic circulation. The objective of the present study was to investigate the effects of excessive barley feeding on the electrophysiological and barrier functions of the ruminal epithelium and serum inflammation and ketogenesis markers after a long-term feeding challenge, using growing goats as a ruminant model. A feeding trial was carried out with growing goats allocated to 1 of the 3 groups (n=5-6 animals/group), with diets consisting exclusively of hay (control diet) or hay with 30 or 60% barley grain. Samples of the ventral ruminal epithelium were taken after euthanasia and instantly subjected to Ussing chamber experiments, where electrophysiological properties of the epithelium were measured in parallel with the permeability of marker molecules of different sizes [fluorescein 5(6)-isothiocyanate and horseradish peroxidase] from luminal to apical side. Additionally, ruminal fluid and blood samples were taken at the beginning of the experiment as well as shortly before euthanasia. Ruminal fluid samples were analyzed for volatile fatty acids and pH, whereas blood samples were analyzed for lipopolysaccharide, serum amyloid A, and β-hydroxybutyrate. Electrophysiological data indicated that barley feeding increased the epithelial short-circuit current compared with the control. Tissue conductance also increased with dietary barley inclusion. As shown with both marker molecules, permeability of ruminal epithelia increased with barley inclusion in the diet. Despite a lowered ruminal pH associated with increased volatile fatty acids (such as propionate and butyrate) concentrations as well as altered epithelial properties in response to high-grain feeding, no signs of inflammation became apparent, as blood serum amyloid A concentrations remained unaffected by diet. However, greater amounts of grain in the diet were associated with a quadratic increase in lipopolysaccharide concentration in the serum. Also, increasing the amounts of barley grain in the diet resulted in a tendency to quadratically augment serum concentrations of β-hydroxybutyrate and, hence, the alimentary ketogenesis. Further studies are needed to clarify the role of barley inclusion in the development of subacute ruminal acidosis in relation to ruminal epithelial damage and the translocation of toxic compounds in vivo. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.