Endoscopic ultrasound imaging has potential for improving the diagnosis of gastrointestinal disease. However, the anatomic correlates of gastrointestinal ultrasound images have not been precisely defined. We have compared ultrasound images with the corresponding histologic sections of 81 specimens of resected and postmortem, normal and diseased gastrointestinal tissue. The five layers seen on ultrasound images of the normal gastrointestinal tract correspond to (1) superficial mucosa, (2) deep mucosa, (3) submucosa plus the acoustical interface between the submucosa and muscularis propria, (4) muscularis propria minus the acoustical interface between the submucosa and muscularis propria, and (5) serosa and subserosal fat. This interpretation takes into consideration the echoes produced by the tissue layers and the echoes produced by the interfaces between layers. Abnormal findings on ultrasound images of neoplastic and inflammatory diseases correspond to histologic tissue structure. When properly interpreted, ultrasound images of the gastrointestinal wall can provide potentially useful diagnostic information.