Ablation of intestinal DMT1 abolished the increase in iron flux induced by exposure to the 4 AA formulation. Duodenal epithelial tissues isolated from DMT1 intestine-specific knockout (DMT1^int/int) mice were mounted in Ussing chambers and bathed in control or 4 AA formulations. Tissues were paired based on conductance, 15 µCi of ⁵⁹Fe was added to one side of the chamber, and samples were acquired every 15 minutes from the other side for 1 hour. J_net was calculated by subtracting J_sm from J_ms. No significant difference was observed with respect to ⁵⁹Fe flux (A) (n = 3-6 per group for J_net) or conductance (B) (n = 6 to 12 per group) in the presence of the control or 4 AA formulations. Data were analyzed by Student’s t test for each sex and at each time point for conductance.