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Intersphincteric resection has become a widely used treatment for patients with rectal cancer. However, the detailed anatomy of the anal canal related to this procedure has remained unclear.The purpose of this study was to clarify the detailed anatomy of the anal canal.This is a descriptive study.Histologic evaluations of paraffin-embedded tissue specimens were conducted at a tertiary referral hospital.Tissue specimens were obtained from cadavers of 5 adults and from 13 patients who underwent abdominoperineal resection for rectal cancer.Sagittal sections from 9 circumferential portions of the cadaveric anal canal (histologic staining) and 3 circumferential portions from patients were studied (immunohistochemistry for smooth and skeletal muscle fibers).Longitudinal fibers between the internal and external anal sphincters consisted primarily of smooth muscle fibers that continued from the longitudinal muscle of the rectum. The levator ani muscle attached directly to the lateral surface of the longitudinal smooth muscle of the rectum. The length of the attachment was longer in the anterolateral portion and shorter in the posterior portion of the anal canal. In the lateral and posterior portions, the levator ani muscle partially overlapped the external anal sphincter; however, there was less overlap in the anterolateral portion. In the posterior portion, thick smooth muscle was present on the surface of the levator ani muscle and it continued to the longitudinal muscle of the rectum.We observed only limited portions in some surgical specimens because of obstruction by tumors.The levator ani muscle attaches directly to the longitudinal muscle of the rectum. The spatial relationship between the smooth and skeletal muscles differed in different portions of the anal canal. For intersphincteric resection, dissection must be performed between the longitudinal muscle of the rectum and the levator ani muscle/external anal sphincter, and the appropriate surgical lines must be selected based on the specific structural characteristics of each portion.