Understanding the association between structure and function is vital before considering surgery involving anal sphincter division. By correlating three-dimensional anal endosonography (AES) and three-dimensional anal canal vector volume manometry (VVM), this study details a method to produce measurements of both sphincter length and pressure leading to identification of the functionally important areas of the anal canal. The aim of this study was to provide combined detailed information on anal canal anatomy and physiology. Twelve males and 12 nulliparous females with no bowel symptoms underwent VVM (using a water-perfused, eight-channel radially arranged catheter) and AES. The synchronization of AES and VVM identified that the majority of rest and squeeze anal pressure is present in the portion of the anal canal covered by both anal sphincters. Nearly, 20% of overall resting anal pressure is produced distal to the caudal termination of the internal anal sphincter. Puborectalis accounts for a significantly greater percentage volume of pressure in females both at rest and when squeezing, though the total volume of pressure is not significantly greater. The majority of resting and squeezing pressure and the least asymmetry, in both sexes, is in the portion of the anal canal covered by external anal sphincter. In females, the external anal sphincter is shorter and a proportionately longer puborectalis accounts for a greater percentage of pressure. Sphincter targeted fistula surgery in females must be performed with special caution. A protective role for puborectalis following obstetric anal sphincter injury is suggested.