The intention of this study was to determine the effects of mucosal osmotic pressure on transport and barrier functions of the rumen epithelium of sheep, which were fed various diets: hay ad libitum, or 600, 1200 or 1800 g day−1 of a supplemented diet plus hay ad libitum. The experiments were conducted by using the conventional Ussing chamber technique. Mucosal osmolarity was adjusted to 300 (control), 375 or 450 mosmol l−1. Feeding of a supplemented diet led to a significant increase of mucosal to serosal Na+ transport and net Na+ transport, probably because of an increase of apical Na+–H+ exchange activity. An increase in mucosal osmotic pressure: (a) reduced net Na+ transport in all feeding groups, the remaining net Na+ transport being higher in tissues of sheep fed a supplemented diet; (b) increased transepithelial tissue conductance, this rise being smallest with a high intake of the supplemented diet; and (c) enhanced the serosal to mucosal Na+ transport in tissues of hay-fed sheep and sheep fed with 600 g day−1 of the supplemented diet, while higher intakes of the supplemented diet (1200 and 1800 g) did not produce any effect. All these changes indicate a diet-dependent adaptation to luminal hypertonicity.