Abstract Introduction: M100240—an acetate thioester of MDL 100,173—a dual angiotensin-converting enzyme (ACE)/neutral endopeptidase (NEP) inhibitor is in Phase II development for cardiovascular conditions. The absorption characteristics of M100240 and MDL 100,173 were evaluated in specific regions of the rat gastrointestinal (GI) tract. Methods: Solutions of M100240 and MDL 100,173 were prepared using mucosal Kreb's solution. Four male Sprague–Dawley rats, fasted before tissue procurement, were sacrificed using 95% CO 2 and 5% O 2. The entire small and large intestine were removed, rinsed in serosal Kreb's solution, and segments mounted onto the Sweetana–Grass diffusion chamber. Test compounds were added to the donor compartment and drug-free oxygenated serosal Kreb's solution to the receiving compartment. The temperature of the chambers was maintained at 37 °C and supplied with 95% O 2/5% CO 2. Samples were removed (0.5 ml) at 0, 30, 60, 90, 120, 180, and 240 min from the serosal side. The volume was maintained with drug-free warm serosal Kreb's solution. Samples were diluted with rabbit plasma and analyzed by liquid chromatography/mass spectrometry. Apparent permeation values [Papp (cm/min)] of M100240 and MDL 100,173 through the duodenum, jejunum, ileum, and colon were determined. Results: The mean Papp of M100240 and MDL l00,173 was highest in the duodenum: 2.29×10 −4±2.40×10 −4 and 1.66×10 −4 cm/min±8.33×10 −5, respectively. The mean Papp was lowest in the colon: 3.61×10 −6±3.44×10 −6 and 1.62×10 −5±3.21×10 −6 cm/min for M100240 and MDL 100,173, respectively. Absorption of MDL 100,173, however, was evident throughout the rat GI tract. Discussion: M100240 and MDL 100,173 are predominantly absorbed from the duodenum in the rat GI tract. MDL 100,173 is also absorbed from the jejunum, ileum, and colon. These results, consistent with data obtained in humans, demonstrate the potential predictive value of the Sweetana–Grass model for site of absorption assessments.