The perineal membrane (PM) is a thick, elastic fiber-rich, smooth muscle-poor membrane extending along the vestibule and lower vaginal wall and embedding the urethrovaginal sphincter and compressor urethrae muscles. To provide a better understanding of the topographical relationship between the PM and the levator ani muscle, we examined histological sections from 15 female cadavers. The composite fibers of the PM were usually continuous with that of a fascia covering the inferior or lateral surface of the levator ani (fascia diaphragmatis pelvis inferior) rather than the endopelvic fascia covering the superior or medial surface of the latter muscle. However, this fascial connection was sometimes interrupted by a venous plexus. The deep transverse perineal muscle was consistently adjacent to the posterolateral aspect of the PM, but whether it extended superficially or deeply to the PM depended on size of the muscle. In contrast to the endopelvic fascia embedding abundant middle-sized nerves (cavernous and sphincter nerves; 0.05-0.1 mm in thickness), the PM contained very thin nerves: many in 10 cadavers but few in 5 cadavers. Most of the nerves seemed to be sensory on the basis of immunohistochemistry. The levator ani muscle was considered likely to provide traction force to the PM, but active elevation appeared to be difficult because of the highly elastic nature of the PM and the interrupting venous plexus. Loss of nerves in the PM might be one of a number of factors that can accelerate pelvic organ prolapse.