Conventional ultrastructural autoradiographic and morphologic studies of the duodenal mucosal cell have generally corroborated physiologic observations of iron absorption, but such methods have limited resolution and fail to distinguish ferric and ferrous iron. This study describes the application of the Prussian blue reaction as an electron microscopic cytochemical stain to the investigation of inorganic iron absorption in iron-deficient, normal, and iron-loaded rats. Ferrous iron is converted to ferric iron at the microvillus membrane. Subsequently intraepithelial ferric iron appears bound to a non-heme acceptor substance in microvilli and later appears as small non-membrane-bound stain deposits which are concentrated in the apical cytoplasm. The appearance of larger stain deposits in the lateral intercellular spaces, in the basal extracellular spaces, and along the intraluminal and extraluminal outer plasmalemma of adjacent endothelial cells of the lamina propria suggests passage of iron from epithelial cells through the lamina propria to blood vessels. The extreme sensitivity of the method compared with simultaneous ultrastructural autoradiographic techniques is demonstrated and suggests usefulness of the method in further studies of iron metabolism.