Our objective was to determine if increasing the ruminal butyrate concentration would improve the selective permeability of ruminal epithelia. Suffolk wether lambs (n = 18) with an initial BW of 47.4 ±1.4 kg were housed in individual pens (1.5 × 1.5 m) with rubber mats on the floor. Lambs were blocked by initial BW into 6 blocks and, within block, were randomly assigned to either the control (CON) or 1 of 2 butyrate supplementation amounts (i.e., 1.25% or 2.50% butyrate as a proportion of DMI). With the exception of butyrate supplementation, all lambs were fed a common diet (90% concentrate and 10% barley silage). After a 14-d feeding period, lambs were killed, and ruminal epithelia from the ventral sac were mounted in Ussing chambers. To facilitate the Ussing chamber measurements, only 1 lamb was killed on an individual day. Thus, the starting date was staggered so that all lambs were exposed to the same experimental protocol. In Ussing chambers, epithelia were incubated using separate mucosal (pH 6.2) and serosal (pH 7.4) bathing solutions. Then 1-14C-butyrate (74 kBq/10 mL) was added to the mucosal side and was used to measure the mucosal-to-serosal flux (J(ms-butyrate)) in 2 consecutive 60-min flux periods with simultaneous measurement of transepithelial conductance (G(t)). During the first (challenge) flux period, the mucosal buffer solution was either acidified to pH 5.2 (ACID) or used as a control (pH 6.2; SHAM). Buffer solutions bathing the epithelia were replaced before the second flux period (recovery). Total ruminal short-chain fatty acid and butyrate concentrations were greater (P = 0.001) in lambs fed 2.50% compared with those fed 0% or 1.25% butyrate. The J(ms-butyrate) was less for lambs fed 1.25% and 2.50% butyrate [3.00 and 3.12 μmol/(cm2·h), respectively] than for CON [3.91 μmol/(cm2· h)]. However, no difference (P = 0.13)was observed for G(t). An ex vivo treatment × flux period interaction was detected (P = 0.003) for J(ms-butyrate), where no differences were present between ACID and SHAM during the challenge period, but the Jms-butyrate was less for ACID than for SHAM during recovery. These results indicate that large increases in the ruminal butyrate concentration decrease the selective permeability of the isolated ruminal epithelia.