The endosonographic anatomy of the anal sphincters was studied by in vivo and in vitro correlation with anatomical dissection. Fourteen fresh anorectal specimens (eight abdominoperineal resection, six post mortem) were scanned in a water-bath with sequential dissection and correlation with the ultrasonographic images. Accurate anatomical identification of the layers during dissection was confirmed histologically. Anal endosonography was performed during operation in 12 patients to validate the in vitro findings. The in vivo study confirmed the in vitro results. Basic endosonographic anatomy has been clarified and inaccuracies in the previous description of the longitudinal muscle and external anal sphincter corrected.