GLY-200 is an oral, non-absorbed polymeric drug designed to non-invasively and reversibly enhance the mucus lining of the proximal small intestine to mimic the effects of metabolic surgery and duodenal exclusion devices. In healthy volunteers and rat models of T2D (GK, ZDF, and DIO), GLY-200 produced reductions in postprandial glucose and bodyweight compared to control. This study aimed to visualize the retained duodenal barrier in rats using appropriate imaging modalities. Methods: Imaging of GLY-200 after gavage administration to fasted Sprague-Dawley rats was performed using Computed Tomography (CT) and an In Vivo Imaging System (IVIS). For CT imaging, GLY-200 was covalently conjugated with diatrizoate; images were obtained after transient isoflurane administration 1, 2, and 4 hours after gavage. For IVIS imaging, GLY-200 was reacted with FITC to produce a fluorescein-GLY-200 conjugate. Rats were euthanized 0.5, 1, 2, 4, and 8 hours after dosing and the GI tracts were immediately removed for imaging. Results: In CT imaging, GLY-200-diatrizoate was retained in the proximal small intestine 1 and 2 hours after administration, with an intense signal only in the duodenum. In comparison, a diatrizoate control was broadly distributed over the small intestine distal to the duodenum by 1 hour. In IVIS imaging, GLY-200-FITC showed intense fluorescence in the proximal small intestine that was predominantly associated with the gut wall and persisted for >4 hours after administration. In comparison, a FITC-dextran70 control was distributed broadly throughout the distal intestine and cecum by 30 minutes with no retention in the duodenum. Conclusion: Imaging studies confirm that GLY-200 conjugates are retained in the duodenum after oral administration. These data support the hypothesis that GLY-200 treatment results in a pharmacologic duodenal exclusion that could potentially recapitulate the glycemic and weight effects of metabolic surgery and duodenal exclusion devices. Disclosure A.Nimgaonkar: Employee; Glyscend Inc. C.Bryant: Employee; Glyscend Inc. T.Carlson: Employee; Glyscend Inc. F.V.Guerina: Employee; Glyscend Inc. K.Colbert: Consultant; LifeSprout, Employee; Glyscend Inc. S.Polomoscanik: None. J.S.Petersen: None. T.H.Jozefiak: Employee; Glyscend Inc., Stock/Shareholder; Glycologix, LLC. M.Fineman: Employee; Glyscend Inc., Stock/Shareholder; Glyscend Inc.