The purpose of this study was to correlate the MRI features of the anal canal with histologic findings. T1- and T2-weighted MR images of nine anal canals were obtained after fixation in 10% formalin. In three specimens, imaging was repeated after removal of histologic layers with a dissecting microscope. Corresponding histologic slices were stained with hematoxylin-eosin, Masson trichrome, and periodic acid-Schiff. Four layers were visualized on T2-weighted images. An inner layer of high signal intensity and a second layer of low signal intensity corresponded to the mucosa as well as mucous secretions and to the submucosa. The high signal intensity layer vanished at the distal part of the anal canal in accordance with the lack of mucus-secreting epithelium below the level of the dentate line. A third layer of intermediate signal intensity corresponded to the internal sphincter. A fourth layer of low signal intensity corresponded to the longitudinal muscle and external sphincter. T2-weighted MRI is capable of showing the internal architecture of the wall of the anal canal. In particular, the internal sphincter can be differentiated from the external sphincter and longitudinal muscle.