Accurate evaluation of atrial septal defect (ASD) size and shape is very important for the selection of patients for transcatheter occlusion. The ability of volume-rendered, three-dimensional echocardiography (3DE) in displaying ASDs in a dynamic mode has been demonstrated; however, its accuracy in sizing ASDs is unknown. To assess this, we performed 3DE of 10 explanted pig hearts in which ASDs of various locations, sizes, and shapes had been experimentally created. From en face 3DE views of the atrial septum containing the defects, major and minor diameters of the defect were measured by a blinded observer, and these data were compared to direct anatomic measurements. The correlations between 3DE and anatomy for the major and minor ASD diameters were y = 0.83x + 3.4 (r = 0.97, p < 0.0001) and y = 0.92x + 1.3 (r = 0.92, p < 0.0001) respectively. The correlation between the measures for major and minor axis ratio was y = 1.06 x - 0.052, r = 0.91, p < 0.0002. Good agreement between both methods of measurements was demonstrated for all measurements. In addition, 3DE portrayed the location and shape of the defects accurately. Thus 3DE provides excellent visualization of ASD and is able to accurately define the size of the defects. These qualitative and quantitative capabilities enhance the clinical potential of this technique in the appraisal of ASDs for decisions regarding application of closure devices.