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There are currently no standardized three-dimensional (3D) transesophageal echocardiographic (TEE) views of the interatrial septum and atrial septal defects (ASDs). Without a standardized approach, it is difficult to ascertain the important anatomic relationships (such as the location of the aortic rim of an ASD), to perform relevant measurements (such as the size of an ASD or the size of its rims), or to guide the deployment of catheters and devices during atrial septal closure.Using a 3D TEE matrix-array transducer, 706 TEE studies were performed over a 14-month period. The purpose of the study was to develop a standardized protocol for anatomically correct orientation of 3D TEE images of the interatrial septum and ASDs.Among 706 TEE studies, there were 23 patients with ASDs, representing 3.3% of the study population. Eighteen patients had secundum ASDs, two had primum ASDs, and three had sinus venosus ASDs of the superior vena cava type. A protocol for properly orienting 3D TEE images of the interatrial septum and ASDs was developed. When the images are acquired at an angle of 0°, the septum is properly oriented by the tilt-up-then-left maneuver. The initial 3D TEE image in first tilted up to reveal the right atrial side of the septum. Then the image is tilted 180° around its vertical axis to reveal the left atrial side of the septum; the aortic rim is on the left, the superior vena cava on the top, and the right-sided pulmonary vein ostia on the right side of the screen. For acquisitions at a higher angle, the rotate-left-in-z-axis maneuver is used. The image is first tilted up to reveal the right atrial side of the septum, as in the tilt-up-then-left maneuver. The image is then rotated counterclockwise in the z axis until the superior vena cave is at 12 o'clock. Finally, the image is tilted 180° around its vertical axis to reveal the left atrial side of the septum.The use of standardized tilt-up-then-left and rotate-left-in-z-axis maneuvers enhances the diagnosis of ASDs, ascertains the important anatomic relationships of ASDs to surrounding structures, and facilitates communication between echocardiographers obtaining 3D TEE images and interventional cardiologists or cardiac surgeons performing ASD closures.