Background: Tramadol is a centrally acting analgesic that is often used in conjunction with nonsteroidal anti-inflammatory drugs (NSAIDs). The effect of coadministration of tramadol and indomethacin on gastric barrier function in dogs is unknown. Hypothesis/Objectives: That coadministration of a nonselective NSAID (indomethacin) and tramadol would decrease recovery of barrier function as compared with acid-injured, indomethacin-treated, and tramadol-treated mucosa. Animals: Gastric mucosa of 10 humanely euthanized shelter dogs. Methods: Ex vivo study. Mounted gastric mucosa was treated with indomethacin, tramadol, or both. Gastric barrier function, prostanoid production, and cyclooxygenase expression were quantified. Results: Indomethacin decreased recovery of transepithelial electrical resistance after injury, although neither tramadol nor the coadministration of the two had an additional effect. Indomethacin inhibited production of gastroprotective prostanoids prostaglandin E2 (acid-injured PGE2: 509.3±158.3pg/mL, indomethacin+acid injury PGE2: 182.9±93.8pg/mL, P<.001) and thromboxane B2 (acid-injured TXB2: 233.2±90.7pg/mL, indomethacin+acid injury TXB2: 37.9±16.8pg/mL, P<.001), whereas tramadol had no significant effect (PGE2 P=.713, TXB2 P=.194). Neither drug had an effect on cyclooxygenase expression (COX-1 P=.743, COX-2 P=.705). Acid injury induced moderate to marked epithelial cell sloughing, which was unchanged by drug administration. Conclusions and Clinical Importance: There was no apparent interaction of tramadol and a nonselective cyclooxygenase in this ex vivo model. These results suggest that if there is an adverse interaction of the 2 drugs in vivo, it is unlikely to be via prostanoid inhibition.