The objective of this study was to identify whether ruminal acidosis (RA) or feed restriction (FR) differentially affect permeability of the gastrointestinal tract (GIT). Twenty-one Holstein steers were randomly assigned to 1 of 3 treatments: control (CON); ruminal acidosis (ACID), and feed restriction (REST). Steers were fed a common diet with a 50:50 F:C ratio once daily at 0800 h for a 5-d baseline period followed by a challenge period. Rumen acidosis was induced by restricting feed to 25% DMI for 1 d and then offering pelleted barley (30% DMI:BW) the next day. Steers on the REST treatment were restricted to 25% DMI for 5 d. Steers were killed and tissues were collected from the rumen, omasum, duodenum, jejunum, ileum, cecum, and proximal and distal colon for measurement of ¹⁴C-mannitol and ³H-inulin flux in Ussing chambers as markers for gut permeability. Data were analyzed as a randomized complete block design using Proc Mixed. Rumen pH was recorded throughout the study. During baseline, there were no differences for DMI or rumen pH (7.1 kg/d and 6.53, respectively; P > 0.1), but DMI and pH during the challenge were 1.7 kg/d and 6.94, and 7.9 kg/d and 5.86 for REST and ACID calves, respectively. The proportion of acetate in the proximal colon from CON (56.8%) was greater (treatment × region, P = 0.01) than for FR (31.0%) and ACID (31.0%). Butyrate concentration was less in the proximal colon of CON (16.0%; interaction P < 0.01) than ACID (31.0%). There were no region × treatment interactions or treatment effects (P > 0.10) for inulin or mannitol flux. However, inulin flux (nmol/(cm² × h)) was greater (P < 0.01) in rumen (21.1), omasum (26.9), and duodenum (21.3) than ileum (4.2), cecum (7.5), and distal colon (2.4). Mannitol flux (nmol/(cm² × h)) was greatest (P < 0.01) in rumen (36.9), duodenum (59.5), jejunum (48.2), and proximal (41.4) and distal colon (33.0) relative to ileum (13.0). These data indicate that feed restriction and ruminal acidosis do not appear to differentially affect permeability of the GIT. The duodenum and rumen are likely regions with greatest permeability.